Innovations in functional genomics and molecular breeding of pea: exploring advances and opportunities.

The garden pea (Pisum sativum L.) is a significant cool-season legume, serving as crucial food sources, animal feed, and industrial raw materials. The advancement of functional genomics over the past two decades has provided substantial theoretical foundations and progress to pea breeding. Notably, the release of the pea reference genome has enhanced our understanding of plant architecture, symbiotic nitrogen fixation (SNF), flowering time, floral organ development, seed development, and stress resistance. However, a considerable gap remains between pea functional genomics and molecular breeding. This review summarizes the current advancements in pea functional genomics and breeding while highlighting the future challenges in pea molecular breeding.

Development of a next generation SNP genotyping array for wheat.

High-throughput genotyping arrays have provided a cost-effective, reliable and interoperable system for genotyping hexaploid wheat and its relatives. Existing, highly cited arrays including our 35K Wheat Breeder's array and the Illumina 90K array were designed based on a limited amount of varietal sequence diversity and with imperfect knowledge of SNP positions. Recent progress in wheat sequencing has given us access to a vast pool of SNP diversity, whilst technological improvements have allowed us to fit significantly more probes onto a 384-well format Axiom array than previously possible. Here we describe a novel Axiom genotyping array, the 'Triticum aestivum Next Generation' array (TaNG), largely derived from whole genome skim sequencing of 204 elite wheat lines and 111 wheat landraces taken from the Watkins 'Core Collection'. We used a novel haplotype optimization approach to select SNPs with the highest combined varietal discrimination and a design iteration step to test and replace SNPs which failed to convert to reliable markers. The final design with 43 372 SNPs contains a combination of haplotype-optimized novel SNPs and legacy cross-platform markers. We show that this design has an improved distribution of SNPs compared to previous arrays and can be used to generate genetic maps with a significantly higher number of distinct bins than our previous array. We also demonstrate the improved performance of TaNGv1.1 for Genome-wide association studies (GWAS) and its utility for Copy Number Variation (CNV) analysis. The array is commercially available with supporting marker annotations and initial genotyping results freely available.

PsNRT2.3 interacts with PsNAR to promote high-affinity nitrate uptake in pea (Pisum sativum L.).

Nitrate, the primary form of nitrogen absorbed by plants, supplies essential compounds for plant growth and development. Peas are frequently used as rotation crops to improve and stabilize soil fertility. However, the determinants of nitrate uptake and transport in peas remain largely unclear, primarily due to the pea genome's complexity and size. In this study, we utilized the complete genomic information of peas to identify three PsNRT2 family genes within the pea genome. We conducted a comprehensive examination of their protein conserved domains, physicochemical properties, gene structure, and phylogenetic evolution, revealing PsNRT2.3 as the potential key gene for high-affinity nitrate transport in peas. Subcellular localization studies indicated that PsNRT2.3 resides on the plasma membrane. Using hairy root transformation, we noted the predominant expression of PsNRT2.3 in the root stele, which is inducible by nitrate. Our experiments involving overexpression and silencing methods further confirmed that PsNRT2.3 plays a key role in enhancing nitrate uptake in peas. Additionally, our work showed that PsNAR could interact with PsNRT2.3, modulating pea nitrate uptake. After silencing PsNAR, even with the normal expression of PsNRT2.3, the ability of peas to absorb nitrate was significantly reduced. In conclusion, this study identifies the high-affinity nitrate transport gene PsNRT2.3 in peas and clarifies its critical role and regulatory network in nitrate transport, contributing to a new understanding of nitrate utilization in peas.

Comparative phylogenomics and phylotranscriptomics provide insights into the genetic complexity of nitrogen-fixing root-nodule symbiosis.

Plant root-nodule symbiosis (RNS) with mutualistic nitrogen-fixing bacteria is restricted to a single clade of angiosperms, the Nitrogen-Fixing Nodulation Clade (NFNC), and is best understood in the legume family. Nodulating species share many commonalities, explained either by divergence from a common ancestor over 100 million years ago or by convergence following independent origins over that same time period. Regardless, comparative analyses of diverse nodulation syndromes can provide insights into constraints on nodulation-what must be acquired or cannot be lost for a functional symbiosis-and the latitude for variation in the symbiosis. However, much remains to be learned about nodulation, especially outside of legumes. Here, we employed a large-scale phylogenomic analysis across 88 species, complemented by 151 RNA-seq libraries, to elucidate the evolution of RNS. Our phylogenomic analyses further emphasize the uniqueness of the transcription factor NIN as a master regulator of nodulation and identify key mutations that affect its function across the NFNC. Comparative transcriptomic assessment revealed nodule-specific upregulated genes across diverse nodulating plants, while also identifying nodule-specific and nitrogen-response genes. Approximately 70% of symbiosis-related genes are highly conserved in the four representative species, whereas defense-related and host-range restriction genes tend to be lineage specific. Our study also identified over 900 000 conserved non-coding elements (CNEs), over 300 000 of which are unique to sampled NFNC species. NFNC-specific CNEs are enriched with the active H3K9ac mark and are correlated with accessible chromatin regions, thus representing a pool of candidate regulatory elements for genes involved in RNS. Collectively, our results provide novel insights into the evolution of nodulation and lay a foundation for engineering of RNS traits in agriculturally important crops.

Plant genome resequencing and population genomics: Current status and future prospects.

Advances in DNA sequencing technology have sparked a genomics revolution, driving breakthroughs in plant genetics and crop breeding. Recently, the focus has shifted from cataloging genetic diversity in plants to exploring their functional significance and delivering beneficial alleles for crop improvement. This transformation has been facilitated by the increasing adoption of whole-genome resequencing. In this review, we summarize the current progress of population-based genome resequencing studies and how these studies affect crop breeding. A total of 187 land plants from 163 countries have been resequenced, comprising 54 413 accessions. As part of resequencing efforts 367 traits have been surveyed and 86 genome-wide association studies have been conducted. Economically important crops, particularly cereals, vegetables, and legumes, have dominated the resequencing efforts, leaving a gap in 49 orders, including Lycopodiales, Liliales, Acorales, Austrobaileyales, and Commelinales. The resequenced germplasm is distributed across diverse geographic locations, providing a global perspective on plant genomics. We highlight genes that have been selected during domestication, or associated with agronomic traits, and form a repository of candidate genes for future research and application. Despite the opportunities for cross-species comparative genomics, many population genomic datasets are not accessible, impeding secondary analyses. We call for a more open and collaborative approach to population genomics that promotes data sharing and encourages contribution-based credit policy. The number of plant genome resequencing studies will continue to rise with the decreasing DNA sequencing costs, coupled with advances in analysis and computational technologies. This expansion, in terms of both scale and quality, holds promise for deeper insights into plant trait genetics and breeding design.

Biomimetic Nanozymes Suppressed Ferroptosis to Ameliorate Doxorubicin-Induced Cardiotoxicity via Synergetic Effect of Antioxidant Stress and GPX4 Restoration.

Mitochondria-dependent ferroptosis plays an important role in the pathogenesis of doxorubicin (DOX)-induced cardiotoxicity (DIC), which remains a clinical challenge due to the lack of effective interventions. Cerium oxide (CeO2), a representative nanozyme, has attracted much attention because of its antioxidant properties. This study evaluated CeO2-based nanozymes for the prevention and treatment of DIC in vitro and in vivo by adding nanoparticles (NPs), which were synthesized by biomineralization, to the culture or giving them to the mice, and the ferroptosis-specific inhibitor ferrostatin-1 (Fer-1) was used as control. The prepared NPs exhibited an excellent antioxidant response and glutathione peroxidase 4 (GPX4)-depended bioregulation, with the additional merits of bio-clearance and long retention in the heart. The experiments showed that NP treatment could significantly reverse myocardial structural and electrical remodeling, and reduce myocardial necrosis. These cardioprotective therapeutic effects were associated with their ability to alleviate oxidative stress, mitochondrial lipid peroxidation, and mitochondrial membrane potential damage, with a superior efficiency to the Fer-1. The study also found that the NPs significantly restored the expression of GPX4 and mitochondrial-associated proteins, thereby restoring mitochondria-dependent ferroptosis. Therefore, the study provides some insights into the role of ferroptosis in DIC. It also shows that CeO2-based nanozymes could be a promising prevention and treatment candidate as a novel cardiomyocyte ferroptosis protector to mitigate DIC and improve prognosis and quality of life in cancer patients.

The Evolution of C4 Photosynthesis in Flaveria (Asteraceae): Insights from the Flaveria linearis Complex.

Flaveria is a leading model for C4 plant evolution due to the presence of a dozen C3-C4 intermediate species, many of which are associated with a phylogenetic complex centered around Flaveria linearis. To investigate C4 evolution in Flaveria, we updated the Flaveria phylogeny and evaluated gas exchange, starch δ13C, and activity of C4 cycle enzymes in 19 Flaveria species and 28 populations within the F. linearis complex. A principal component analysis identified six functional clusters: (1) C3, (2) sub-C2, (3) full C2, (4) enriched C2, (5) sub-C4, and (6) fully C4 species. The sub-C2 species lacked a functional C4 cycle, while a gradient was present in the C2 clusters from little to modest C4 cycle activity as indicated by δ13C and enzyme activities. Three Yucatan populations of F. linearis had photosynthetic CO2 compensation points equivalent to C4 plants but showed little evidence for an enhanced C4 cycle, indicating they have an optimized C2 pathway that recaptures all photorespired CO2 in the bundle sheath (BS) tissue. All C2 species had enhanced aspartate aminotransferase activity relative to C3 species and most had enhanced alanine aminotransferase activity. These aminotransferases form aspartate and alanine from glutamate and in doing so could help return photorespiratory nitrogen (N) from BS to mesophyll cells, preventing glutamate feedback onto photorespiratory N assimilation. Their use requires upregulation of parts of the C4 metabolic cycle to generate carbon skeletons to sustain N return to the mesophyll, and thus could facilitate the evolution of the full C4 photosynthetic pathway.

The genome of homosporous maidenhair fern sheds light on the euphyllophyte evolution and defences.

Euphyllophytes encompass almost all extant plants, including two sister clades, ferns and seed plants. Decoding genomes of ferns is the key to deep insight into the origin of euphyllophytes and the evolution of seed plants. Here we report a chromosome-level genome assembly of Adiantum capillus-veneris L., a model homosporous fern. This fern genome comprises 30 pseudochromosomes with a size of 4.8-gigabase and a contig N50 length of 16.22 Mb. Gene co-expression network analysis uncovered that homospore development in ferns has relatively high genetic similarities with that of the pollen in seed plants. Analysing fern defence response expands understanding of evolution and diversity in endogenous bioactive jasmonates in plants. Moreover, comparing fern genomes with those of other land plants reveals changes in gene families important for the evolutionary novelties within the euphyllophyte clade. These results lay a foundation for studies on fern genome evolution and function, as well as the origin and evolution of euphyllophytes.

HAPPE: A Tool for Population Haplotype Analysis and Visualization in Editable Excel Tables.

Haplotype identification, characterization and visualization are important for large-scale analysis and use in population genomics. Many tools have been developed to visualize haplotypes, but it is challenging to display both the pattern of haplotypes and the genotypes for each single SNP in the context of a large amount of genomic data. Here, we describe the tool HAPPE, which uses the agglomerative hierarchical clustering algorithm to characterize and visualize the genotypes and haplotypes in a phylogenetic context. The tool displays the plots by coloring the cells and/or their borders in Excel tables for any given gene and genomic region of interest. HAPPE facilitates informative displays wherein data in plots are easy to read and access. It allows parallel display of several lines of values, such as phylogenetic trees, P values of GWAS, the entry of genes or SNPs, and the sequencing depth at each position. These features are informative for the detection of insertion/deletions or copy number variations. Overall, HAPPE provides editable plots consisting of cells in Excel tables, which are user-friendly to non-programmers. This pipeline is coded in Python and is available at https://github.com/fengcong3/HAPPE.

Three-nucleotide periodicity of nucleotide diversity in a population enables the identification of open reading frames.

Accurate prediction of open reading frames (ORFs) is important for studying and using genome sequences. Ribosomes move along mRNA strands with a step of three nucleotides and datasets carrying this information can be used to predict ORFs. The ribosome-protected footprints (RPFs) feature a significant 3-nt periodicity on mRNAs and are powerful in predicting translating ORFs, including small ORFs (sORFs), but the application of RPFs is limited because they are too short to be accurately mapped in complex genomes. In this study, we found a significant 3-nt periodicity in the datasets of populational genomic variants in coding sequences, in which the nucleotide diversity increases every three nucleotides. We suggest that this feature can be used to predict ORFs and develop the Python package 'OrfPP', which recovers ~83% of the annotated ORFs in the tested genomes on average, independent of the population sizes and the complexity of the genomes. The novel ORFs, including sORFs, identified from single-nucleotide polymorphisms are supported by protein mass spectrometry evidence comparable to that of the annotated ORFs. The application of OrfPP to tetraploid cotton and hexaploid wheat genomes successfully identified 76.17% and 87.43% of the annotated ORFs in the genomes, respectively, as well as 4704 sORFs, including 1182 upstream and 2110 downstream ORFs in cotton and 5025 sORFs, including 232 upstream and 234 downstream ORFs in wheat. Overall, we propose an alternative and supplementary approach for ORF prediction that can extend the studies of sORFs to more complex genomes.

Wheat Panache: A pangenome graph database representing presence-absence variation across sixteen bread wheat genomes.

Bread wheat (Triticum aestivum L.) is one of humanity's most important staple crops, characterized by a large and complex genome with a high level of gene presence-absence variation (PAV) between cultivars, hampering genomic approaches for crop improvement. With the growing global population and the increasing impact of climate change on crop yield, there is an urgent need to apply genomic approaches to accelerate wheat breeding. With recent advances in DNA sequencing technology, a growing number of high-quality reference genomes are becoming available, reflecting the genetic content of a diverse range of cultivars. However, information on the presence or absence of genomic regions has been hard to visualize and interrogate because of the size of these genomes and the lack of suitable bioinformatics tools. To address this limitation, we have produced a wheat pangenome graph maintained within an online database to facilitate interrogation and comparison of wheat cultivar genomes. The database allows users to visualize regions of the pangenome to assess PAV between bread wheat genomes.

Graph pangenomes represent more genomic variants than reference genomes. We present a wheat graph pangenome based on 16 public assemblies. We present Wheat Panache, an online visual representation of this graph. Wheat Panache lets users search the graph for presence-absence variants. We also distribute the graph preindexed for Giraffe utilization.

Dynamic Diversity of NLR Genes in Triticum and Mining of Promising NLR Alleles for Disease Resistance.

Bread wheat is an essential crop with the second-highest global production after maize. Currently, wheat diseases are a serious threat to wheat production. Therefore, efficient breeding for disease resistance is extremely urgent in modern wheat. Here, we identified 2012 NLR genes from hexaploid wheat, and Ks values of paired syntenic NLRs showed a significant peak at 3.1-6.3 MYA, which exactly coincided with the first hybridization event between A and B genome lineages at ~5.5 MYA. We provided a landscape of dynamic diversity of NLRs from Triticum and Aegilops and found that NLR genes have higher diversity in wild progenitors and relatives. Further, most NLRs had opposite diversity patterns between genic and 2 Kb-promoter regions, which might respectively link sub/neofunctionalization and loss of duplicated NLR genes. Additionally, we identified an alien introgression of chromosome 4A in tetraploid emmer wheat, which was similar to that in hexaploid wheat. Transcriptome data from four experiments of wheat disease resistance helped to profile the expression pattern of NLR genes and identified promising NLRs involved in broad-spectrum disease resistance. Our study provided insights into the diversity evolution of NLR genes and identified beneficial NLRs to deploy into modern wheat in future wheat disease-resistance breeding.

Cation and Anion Channelrhodopsins: Sequence Motifs and Taxonomic Distribution.

Cation and anion channelrhodopsins (CCRs and ACRs, respectively) primarily from two algal species, Chlamydomonas reinhardtii and Guillardia theta, have become widely used as optogenetic tools to control cell membrane potential with light. We mined algal and other protist polynucleotide sequencing projects and metagenomic samples to identify 75 channelrhodopsin homologs from four channelrhodopsin families, including one revealed in dinoflagellates in this study. We carried out electrophysiological analysis of 33 natural channelrhodopsin variants from different phylogenetic lineages and 10 metagenomic homologs in search of sequence determinants of ion selectivity, photocurrent desensitization, and spectral tuning in channelrhodopsins. Our results show that association of a reduced number of glutamates near the conductance path with anion selectivity depends on a wider protein context, because prasinophyte homologs with a glutamate pattern identical to that in cryptophyte ACRs are cation selective. Desensitization is also broadly context dependent, as in one branch of stramenopile ACRs and their metagenomic homologs, its extent roughly correlates with phylogenetic relationship of their sequences. Regarding spectral tuning, we identified two prasinophyte CCRs with red-shifted spectra to 585 nm. They exhibit a third residue pattern in their retinal-binding pockets distinctly different from those of the only two types of red-shifted channelrhodopsins known (i.e., the CCR Chrimson and RubyACRs). In cryptophyte ACRs we identified three specific residue positions in the retinal-binding pocket that define the wavelength of their spectral maxima. Lastly, we found that dinoflagellate rhodopsins with a TCP motif in the third transmembrane helix and a metagenomic homolog exhibit channel activity. IMPORTANCE Channelrhodopsins are widely used in neuroscience and cardiology as research tools and are considered prospective therapeutics, but their natural diversity and mechanisms remain poorly characterized. Genomic and metagenomic sequencing projects are producing an ever-increasing wealth of data, whereas biophysical characterization of the encoded proteins lags behind. In this study, we used manual and automated patch clamp recording of representative members of four channelrhodopsin families, including a family in dinoflagellates that we report in this study. Our results contribute to a better understanding of molecular determinants of ionic selectivity, photocurrent desensitization, and spectral tuning in channelrhodopsins.

An anchored chromosome-scale genome assembly of spinach improves annotation and reveals extensive gene rearrangements in euasterids.

Spinach (Spinacia oleracea L.) is a member of the Caryophyllales family, a basal eudicot asterid that consists of sugar beet (Beta vulgaris L. subsp. vulgaris), quinoa (Chenopodium quinoa Willd.), and amaranth (Amaranthus hypochondriacus L.). With the introduction of baby leaf types, spinach has become a staple food in many homes. Production issues focus on yield, nitrogen-use efficiency and resistance to downy mildew (Peronospora effusa). Although genomes are available for the above species, a chromosome-level assembly exists only for quinoa, allowing for proper annotation and structural analyses to enhance crop improvement. We independently assembled and annotated genomes of the cultivar Viroflay using short-read strategy (Illumina) and long-read strategies (Pacific Biosciences) to develop a chromosome-level, genetically anchored assembly for spinach. Scaffold N50 for the Illumina assembly was 389 kb, whereas that for Pacific BioSciences was 4.43 Mb, representing 911 Mb (93% of the genome) in 221 scaffolds, 80% of which are anchored and oriented on a sequence-based genetic map, also described within this work. The two assemblies were 99.5% collinear. Independent annotation of the two assemblies with the same comprehensive transcriptome dataset show that the quality of the assembly directly affects the annotation with significantly more genes predicted (26,862 vs. 34,877) in the long-read assembly. Analysis of resistance genes confirms a bias in resistant gene motifs more typical of monocots. Evolutionary analysis indicates that Spinacia is a paleohexaploid with a whole-genome triplication followed by extensive gene rearrangements identified in this work. Diversity analysis of 75 lines indicate that variation in genes is ample for hypothesis-driven, genomic-assisted breeding enabled by this work.

Giant DNA viruses make big strides in eukaryote evolution.

Nucleocytoplasmic large DNA viruses (NCLDVs) are widespread in the biosphere. This issue of Cell Host & Microbe, Nelson et al., and a recent Nature paper, Moniruzzaman et al., show NCLDVs can integrate into host genomes, highlighting a mechanism of large-scale virus-mediated horizontal gene transfer (vHGT) driving eukaryotic evolution.

Genetic insights into the evolution of genera with the eastern Asia-eastern North America floristic disjunction: a transcriptomics analysis.

PREMISE: Large disjunctions in species distributions provide excellent opportunities to study processes that shape biogeographic patterns. One such disjunction is the eastern Asia-eastern North America (EA-ENA) floristic disjunction. For many genera with this disjunction, species richness is greater in EA than in ENA; this pattern has been attributed, in part, to higher rates of molecular evolution and speciation in EA. Longer branch lengths have been found in some EA clades, relative to their ENA sister clades, suggesting that the EA lineages have evolved at a higher rate, possibly due to environmental heterogeneity, potentially contributing to the species richness anomaly. METHODS: To evaluate whether rates of molecular evolution are elevated in EA relative to ENA, we used transcriptomes from species in 11 genera displaying this disjunction. Rates of molecular evolution were estimated for up to 385 orthologous nuclear loci per genus. RESULTS: No statistically significant differences were identified in pairwise comparisons between EA and ENA sister species, suggesting equal rates of molecular evolution for both species; the data also suggest similar selection pressures in both regions. For larger genera, evidence likewise argues against more species-rich clades having higher molecular evolutionary rates, regardless of region. Our results suggest that genes across multiple gene ontology categories are evolving at similar rates under purifying selection in species in both regions. CONCLUSIONS: Our data support the hypothesis that greater species richness in EA than ENA is due to factors other than an overall increase in rates of molecular evolution in EA.

Author Correction: The genome of Prasinoderma coloniale unveils the existence of a third phylum within green plants.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The genome of Prasinoderma coloniale unveils the existence of a third phylum within green plants.

Genome analysis of the pico-eukaryotic marine green alga Prasinoderma coloniale CCMP 1413 unveils the existence of a novel phylum within green plants (Viridiplantae), the Prasinodermophyta, which diverged before the split of Chlorophyta and Streptophyta. Structural features of the genome and gene family comparisons revealed an intermediate position of the P. coloniale genome (25.3 Mb) between the extremely compact, small genomes of picoplanktonic Mamiellophyceae (Chlorophyta) and the larger, more complex genomes of early-diverging streptophyte algae. Reconstruction of the minimal core genome of Viridiplantae allowed identification of an ancestral toolkit of transcription factors and flagellar proteins. Adaptations of P. coloniale to its deep-water, oligotrophic environment involved expansion of light-harvesting proteins, reduction of early light-induced proteins, evolution of a distinct type of C4 photosynthesis and carbon-concentrating mechanism, synthesis of the metal-complexing metabolite picolinic acid, and vitamin B1, B7 and B12 auxotrophy. The P. coloniale genome provides first insights into the dawn of green plant evolution.