Biology subjects

Schranz, M. E.

Publications and source records attributed to Schranz, M. E..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

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. It is widely accepted that nodulation originated through the assembly of modules recruited from existing functions, such as mycorrhizal symbiosis, polar growth, and lateral root development. Because nodulating species are scattered within the NFNC, the number of times nodulation has evolved or has been lost has been a matter of considerable speculation. This interesting evolutionary question has practical implications concerning the ease with which nodulation might be engineered in non-nodulating crop plants. Nodulating species share many commonalities, due either to divergence from a common ancestor over 100 million years ago or to convergence or deep homology following independent origins over that same time period. In either case, 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 what the latitude is for variation in the symbiosis. However, much remains to be learned about nodulation, especially outside of legumes. Here we present new information across the spectrum of nodulating groups. We find no evidence for convergence at the level of amino acid residues or gene family expansion across the NFNC. Our phylogenomic analyses further emphasize the uniqueness of the transcription factor, NIN, as a master regulator of nodulation, and identify key mutations affecting its function across the NFNC. We find that nodulation genes are over-represented among orthologous gene groups (OGs) present in the NFNC common ancestor, but that lineage-specific OGs play major roles in nodulation. We identified over 900,000 conserved noncoding elements (CNEs), of which over 300,000 were unique to NFNC species. A significant proportion of these are associated with nodulation-related genes and thus are candidates for transcriptional regulators.

evolutionary biology↗

bioRxiv2023

A rapid method to quantify vein density in C4 plants using starch staining

C4 photosynthesis has evolved multiple times in the angiosperms and typically involves alterations to the biochemistry, cell biology and development of leaves. One common modification found in C4 plants compared with the ancestral C3 state is an increase in vein density such that the leaf contains a larger proportion of bundle sheath cells. Recent findings indicate that there may be significant intra-specific variation in traits such as vein density in C4 plants but to use such natural variation for trait-mapping, rapid phenotyping would be required. Here we report a high-throughput method to quantify vein density that leverages the bundle sheath specific accumulation of starch found in C4 species. Starch staining allowed high-contrast images to be acquired that permitted image analysis using a MATLAB-based program. The method works for the dicotyledon Gynandropsis gynandra where significant variation in vein density was detected between natural accessions, and the monocotyledon Zea mays where no variation was apparent in the genotypically diverse lines assessed. We anticipate this approach will be useful to map genes controlling vein density in C4 species demonstrating natural variation for this trait. One sentence summaryPreferential accumulation of starch in bundle sheath cells of C4 plants allows high-throughput phenotyping of vein density.

plant biology↗

bioRxiv2022

The genome of Lactuca saligna, a wild relative of lettuce, provides insight into non-host resistance to the downy mildew Bremia lactucae

Lactuca saligna L. is a wild relative of cultivated lettuce (Lactuca sativa L.), with which it is partially interfertile. Hybrid progeny suffer from hybrid incompatibilities (HI), resulting in reduced fertility and distorted transmission ratios. Lactuca saligna displays broad spectrum resistance against lettuce downy mildew caused by Bremia lactucae Regel and is considered a non-host species. This phenomenon of resistance in L. saligna is called non-host resistance (NHR). One possible mechanism behind this NHR is through the plant-pathogen interaction triggered by pathogen-recognition receptors, including nucleotide-binding leucin-rich repeats (NLRs) and receptor-like kinases (RLKs). We report a chromosome-level genome assembly of L. saligna (accession CGN05327), leading to the identification of two large paracentric inversions (>50 Mb) between L. saligna and L. sativa. Genome-wide searches delineated the major resistance clusters as regions enriched in NLRs and RLKs. Three of the enriched regions co-locate with previously identified NHR intervals. RNA-seq analysis of Bremia infected lettuce identified several differentially expressed RLKs in NHR regions. Three tandem wall-associated kinase-encoding genes (WAKs) in the NHR8 interval display particularly high expression changes at an early stage of infection. We propose RLKs as strong candidate(s) for determinants for the NHR phenotype of L. saligna.

genomics↗

bioRxiv2022

Global genomic analysis reveals the genetic origin and secondary invasion of fall armyworm in the Eastern hemisphere

The major plant pest fall armyworm (FAW), Spodoptera frugiperda, is native to the Americas and has colonized African and Asian countries in the Eastern hemisphere since 2016, causing severe damage to multiple agricultural crop species. However, the genetic origin of these invasive populations require more in-depth exploration. We analyzed genetic variation across FAW genomes of 153 newly sequenced individuals from Eastern hemisphere and 127 individuals mostly originating from the Americas. The global genetic structure of FAW shows that the FAW in American has experienced deep differentiation, largely consistent with the Z-chromosomal Tpi haplotypes commonly used to differentiate "corn-strain" and "rice-strain" populations. Results indicate that the invasive Eastern hemisphere populations are different from the American ones and have relatively homogeneous population structure, consistent with the common origin and recent spreading from Africa to Asia. Our analyses suggest that north-and central American "corn-strain" FAW are the most likely sources of the invasion into the Eastern hemisphere. Furthermore, evidence based on genomic, transcriptomic and mitochondrial haplotype network analysis suggest that there has been an earlier independent introduction of FAW into Africa that introgressed into the recent invasive population.

molecular biology↗

bioRxiv2022

Synteny identifies reliable orthologs for phylogenomics and comparative genomics of the Brassicaceae

Large genomic datasets are becoming the new normal for any facet of phylogenetic research, but identification of true orthologous genes and exclusion of problematic paralogs is still challenging when applying commonly used sequencing methods such as target enrichment. Here, we compared conventional ortholog detection using OrthoFinder with ortholog detection through genomic synteny in a dataset of eleven representative diploid Brassicaceae whole genome sequences spanning the entire phylogenetic space. We then evaluated the resulting gene sets regarding gene number, functional annotation, gene and species tree resolution. Finally, we used the syntenic gene sets for comparative genomics and ancestral genome analysis. The use of synteny resulted in considerably more orthologs and also allowed us to reliably identify paralogs. Surprisingly, we did not detect notable differences between species trees reconstructed from syntenic orthologs compared other gene sets, including the Angiosperm353 set and a Brassicaceae specific target enrichment gene set. However, the synteny dataset comprised a multitude of gene functions, strongly suggesting that this method of marker selection for phylogenomics is suitable for studies that value downstream gene function analysis, gene interaction and network studies. Finally, we present the first ancestral genome reconstruction for the Core Brassicaceae predating the Brassicaceae lineage diversification [~]25 million years ago.

evolutionary biology↗

bioRxiv2022

The genome of Gynandropsis gynandra provides insights into whole-genome duplications and the evolution of C4 photosynthesis in Cleomaceae

Gynandropsis gynandra (Cleomaceae) is a cosmopolitan leafy vegetable and medicinal plant, which has also been used as a model to study C4 photosynthesis due to its evolutionary proximity to Arabidopsis. Here, we present a high-quality genome sequence of G. gynandra, anchored onto 17 main super- scaffolds with a total length of 740 Mb, an N50 of 42 Mb and 30,933 well-supported gene models. The G. gynandra genome and previously released genomes of C3 relatives in the Cleomaceae and Brassicaceae make an excellent model for studying the role of genome evolution in the transition from C3 to C4 photosynthesis. We revealed that G. gynandra and its C3 relative Tarenaya hassleriana shared a whole-genome duplication event (Gg-), then an addition of a third genome (Th-, +1x) took place in T. hassleriana but not in G. gynandra. Analysis of syntenic copy number of C4 photosynthesis-related gene families indicates that G. gynandra generally retained more duplicated copies of these genes than C3 T. hassleriana, and also that the G. gynandra C4 genes might have been under positive selection pressure. Both whole-genome and single-gene duplication were found to contribute to the expansion of the aforementioned gene families in G. gynandra. Collectively, this study enhances our understanding of the impact of gene duplication and gene retention on the evolution of C4 photosynthesis in Cleomaceae.

plant biology↗

bioRxiv2022

GENESPACE: syntenic pan-genome annotations for eukaryotes

The development of multiple high-quality reference genome sequences in many taxonomic groups has yielded a high-resolution view of the patterns and processes of molecular evolution. Nonetheless, leveraging information across multiple reference haplotypes remains a significant challenge in nearly all eukaryotic systems. These challenges range from studying the evolution of chromosome structure, to finding candidate genes for quantitative trait loci, to testing hypotheses about speciation and adaptation in nature. Here, we address these challenges through the concept of a pan-genome annotation, where conserved gene order is used to restrict gene families and define the expected physical position of all genes that share a common ancestor among multiple genome annotations. By leveraging pan-genome annotations and exploring the underlying syntenic relationships among genomes, we dissect presence-absence and structural variation at four levels of biological organization: among three tetraploid cotton species, across 300 million years of vertebrate sex chromosome evolution, across the diversity of the Poaceae (grass) plant family, and among 26 maize cultivars. The methods to build and visualize syntenic pan-genome annotations in the GENESPACE R package offer a significant addition to existing gene family and synteny programs, especially in polyploid, outbred and other complex genomes.

genomics↗

bioRxiv2022

The genome sequence of Hirschfeldia incana, a species with high photosynthetic light-use efficiency

Photosynthesis is a biophysical and biochemical process that plays a key role in sustaining plant and human life, being the first step in the production of energy-rich molecules and oxygen in the biosphere. Improving the photosynthetic capacity of agricultural crops is highly desirable to increase their yields. While the core mechanisms of photosynthesis are highly conserved, certainly in higher plants, plants that can maintain a high photosynthetic light-use efficiency at high irradiance are exceptional and may be useful to understand and improve high irradiance photosynthesis of crops. One such exceptional species is Hirschfeldia incana, a member of the well-studied Brassicaceae family that is easy to grow under standard laboratory conditions, providing an excellent resource for studying the genetic and physiological basis of this trait. Here, we present a reference assembly of H. incana and affirm its high photosynthetic efficiency relative to the Brassicaceae species Brassica rapa, Brassica nigra, and Arabidopsis thaliana. We estimate that it diverged from B. rapa and B. nigra 10-11 million years ago and that its genome has diversified from that of the latter two species through large chromosomal rearrangements, species-specific transposon activity, and differential retention of duplicated genes. Genes present at copy numbers different from B. rapa and B. nigra include those involved in photosynthesis and/or abiotic stress, which may mediate the high photosynthetic efficiency of H. incana. We expect the reference assembly of H. incana to be a valuable genomic resource for identifying ways to enhance photosynthetic rates in crop species.

bioinformatics↗

bioRxiv2021

Genome and transcriptome analysis of the beet armyworm Spodoptera exigua reveals targets for pest control

BackgroundThe genus Spodoptera (Lepidoptera: Noctuidae) includes some of the most infamous insect pests of cultivated plants including Spodoptera frugiperda, Spodoptera litura and Spodoptera exigua. To effectively develop targeted pest control strategies for diverse Spodoptera species, genomic resources are highly desired. To this aim, we provide the genome assembly and developmental transcriptome comprising all major life stages of S. exigua, the beet armyworm. Spodoptera exigua is a polyphagous herbivore that can feed from > 130 host plants including several economically important crops. ResultsThe 419 Mb beet armyworm genome was sequenced from a female S. exigua pupa. Using a hybrid genome sequencing approach (Nanopore long read data and Illumina short read), a high-quality genome assembly was achieved (N50=1.1 Mb). An official gene set (OGS, 18,477 transcripts) was generated by automatic annotation and by using transcriptomic RNA-seq data sets of 18 S. exigua samples as supporting evidence. In-depth analyses of developmental stage-specific expression in combination with gene tree analyses of identified homologous genes across Lepidoptera genomes revealed potential Spodoptera-specific genes of interest such as mg7 and REPAT46 upregulated during 1st and 3rd instar larval stages for targeted pest-outbreak management. ConclusionsThe beet armyworm genome sequence and developmental transcriptome covering all major developmental stages provides critical insights into the biology of this devastating polyphagous insect pest species with a worldwide distribution. In addition, comparative genomic analyses across Lepidoptera significantly advance our knowledge to further control other invasive Spodoptera species and reveals potential lineage-specific target genes for pest control strategies.

genomics↗

bioRxiv2021

Deciphering Brassica plant defence responses to cabbage white butterfly egg-associated molecular patterns

Brassica plants activate a strong hypersensitive response (HR)-like necrosis underneath eggs of cabbage white butterflies, but their molecular response to eggs is poorly understood. Here, we developed a method to generate egg wash to identify potential insect egg-associated molecular patterns (EAMPs) inducing HR-like necrosis. We found that egg wash, containing compounds from Pieris eggs, induced a similar response as eggs. We show that wash of hatched eggs, of egg glue, and of accessory reproductive glands (ARG) that produce this glue, also induced HR-like necrosis, whereas removal of the glue from eggs resulted in a reduced response. Eggs of Pieris butterflies induced callose deposition, production of reactive oxygen species and cell death in B. nigra and B. rapa leaf tissue, also in plants that did not express HR-like necrosis. Finally, only washes from Pieris eggs induced defence genes and ethylene production, whereas egg wash of a generalist moth did not. Our results indicate that EAMPs are in the egg glue and that the response in B. nigra is specific to Pieris species. Our study expands knowledge on the Brassica-Pieris-egg interaction, and paves the way for identification of EAMPs in Pieris egg glue and corresponding receptor in Brassica spp.

plant biology↗

bioRxiv2020

Origin and evolution of the cannabinoid oxidocyclase gene family

Cannabis is an ancient crop representing a rapidly increasing legal market, especially for medicinal purposes. Medicinal and psychoactive effects of Cannabis rely on specific terpenophenolic ligands named cannabinoids. Recent whole-genome sequencing efforts have uncovered variation in multiple genes encoding the final steps in cannabinoid biosynthesis. However, the origin, evolution, and phylogenetic relationships of these cannabinoid oxidocyclase genes remain unclear. To elucidate these aspects we performed comparative genomic analyses of Cannabis, related genera within the Cannabaceae family, and selected outgroup species. Results show that cannabinoid oxidocyclase genes originated in the Cannabis lineage from within a larger gene expansion in the Cannabaceae family. Localization and divergence of oxidocyclase genes in the Cannabis genome revealed two main syntenic blocks, each comprising tandemly repeated cannabinoid oxidocyclase genes. By comparing these blocks with those in genomes from closely related species we propose an evolutionary model for the origin, neofunctionalization, duplication, and diversification of cannabinoid oxidocycloase genes. Based on phylogenetic meta-analyses, we propose a comprehensive classification of three main clades and seven subclades that is intended to aid unequivocal referencing and identification of cannabinoid oxidocyclase genes. Our data suggest that cannabinoid oxidocyclase gene copy number variation may have less functional relevance than previously thought. Instead, we propose that cannabinoid phenotype is primarily determined by presence/absence of single-copy genes. Increased sampling across Cannabis native geographic range is likely to uncover additional cannabinoid oxidocyclase gene sequence variation. Significance statementCannabis genome sequencing efforts have revealed extensive cannabinoid oxidocyclase gene variation. However, phylogenetic relationships and evolution of these genes remains unclear. Our meta analysis of currently available data reveals that these genes comprise three main clades and seven subclades that originated through Cannabis-specific gene duplication and divergence. Our new conceptual and evolutionary framework serves as a reference for future description and functional analyses of cannabinoid oxidocyclases.

evolutionary biology↗

bioRxiv2020

Surprising amount of stasis in repetitive genome content across the Brassicales

Genome size of plants has long piqued the interest of researchers due to the vast differences among organisms. However, the mechanisms that drive size differences have yet to be fully understood. Two important contributing factors to genome size are expansions of repetitive elements, such as transposable elements (TEs), and whole-genome duplications (WGD). Although studies have found correlations between genome size and both TE abundance and polyploidy, these studies typically test for these patterns within a genus or species. The plant order Brassicales provides an excellent system to test if genome size evolution patterns are consistent across larger time scales, as there are numerous WGDs. This order is also home to one of the smallest plant genomes, Arabidopsis thaliana - chosen as the model plant system for this reason - as well as to species with very large genomes. With new methods that allow for TE characterization from low-coverage genome shotgun data and 71 taxa across the Brassicales, we find no correlation between genome size and TE content, and more surprisingly we identify no significant changes to TE landscape following WGD.

evolutionary biology↗

bioRxiv2019

Phylogenomic analysis of the APETALA2 transcription factor subfamily across angiosperms reveals both deep conservation and lineage-specific patterns

The APETALA2 (AP2) subfamily of transcription factors are key regulators of angiosperm root, shoot, flower, and embryo development. The broad diversity of anatomical and morphological structures is potentially associated with the genomic dynamics of the AP2 subfamily. However, a comprehensive phylogenomic analysis of the AP2 subfamily across angiosperms is lacking. We combined phylogenetic and synteny analysis of distinct AP2 subclades in the completed genomes of 107 angiosperm species. We identified major changes in copy number variation and genomic context within subclades across lineages, and discuss how these changes may have contributed to the evolution of lineage-specific traits. Multiple AP2 subclades show highly conserved patterns of copy number and synteny across angiosperms, while others are more dynamic and show distinct lineage-specific patterns. As examples of lineage-specific morphological divergence due to AP2 subclade dynamics, we hypothesize that that loss of PLETHORA1/2 in monocots correlates with the absence of taproots, whereas independent lineage-specific changes of PLETHORA4/BABY BOOM and WRINKLED1 genes in Brassicaceae and monocots point towards regulatory divergence of embryogenesis between these lineages. Additionally, copy number expansion of TOE1 and TOE3/AP2 in asterids is implicated with differential regulation of flower development. Moreover, we show that the genomic context of AP2s is in general highly specialised per angiosperm lineage. Our study is the first to shed light on the evolutionary divergence of the AP2 subfamily subclades across major angiosperm lineages and emphasises the need for lineage-specific characterisation of developmental networks to further understand trait variability. SIGNIFICANCE STATEMENTAPETALA2 transcription factors are crucial regulators of embryogenesis and post-embryonic development in plants. Characterising the genomic dynamics of APETALA2 genes across 107 angiosperms provided evolutionary insights into gene-family and morphological divergence across major angiosperm lineages.

plant biology↗

bioRxiv2019

Insect egg-killing: a new front on the evolutionary arms-race between Brassicaceae plants and Pierid butterflies

Evolutionary arms-races between plants and herbivores have been proposed to generate key innovations that can drive diversification of the interacting species. Recent studies reveal that plant traits that target herbivore insect eggs are widespread throughout the plant kingdom. Within the Brassicaceae family, some plants express a hypersensitive response (HR)-like necrosis underneath the eggs of specialist cabbage white butterflies (Pieridae) that leads to eggs desiccating or dropping of the leaf. Here, we studied the evolutionary basis of this trait, its egg-killing effect on and elicitation by specialist butterflies, by screening 31 Brassicaceae species and nine Pieridae species. We show that induction of HR-like necrosis by pierid egg deposition is clade-specific in the economically important Brassiceae tribe (Brassica crops and close-relatives) and in the first-branching genus Aethionema. The necrosis is elicited only by pierid butterflies that feed on Brassicaceae plants; four Pieris and Anthocharis cardamines butterflies, of which the larvae are specialists on Brassicaceae, elicited a HR-like necrosis. Eggs of pierid butterflies that feed on Rhamnaceae (Gonepteryx rhamni) or Fabaceae (Colias spp.) however, did not elicit such a leaf necrosis. Finally, eggs of Aglais io, a species of the sister group Nymphalidae, did not elicit any visible response. Counter-adaptations to HR-like necrosis might have evolved by insect deposition of eggs in clusters or on inflorescences. Our findings suggest that the plants egg-killing trait is a new front on the evolutionary arms-race between Brassicaceae and pierid butterflies beyond the well-studied chemical defence traits against caterpillars.

evolutionary biology↗

bioRxiv2019

Pangenomic read mapping

In modern genomics, mapping reads to a single reference genome is common practice. However, a reference genome does not necessarily accurately represent a population or species and as a result a substantial percentage of reads often cannot be mapped. A number of graph-based variation-aware mapping methods have recently been proposed to remedy this. Here, we propose an alternative multi-reference approach, which aligns reads to large collections of genomes simultaneously. Our approach, an extension to our pangenomics suite PanTools (https://git.wur.nl/bioinformatics/pantools), is as accurate as state-of the-art tools but more efficient on large numbers of genomes. We successfully applied PanTools to map genomic and metagenomic reads to large collections of viral, archaeal, bacterial, fungal and plant genomes.

bioinformatics↗

bioRxiv2019

Phylogeny and Multiple Independent Whole-Genome Duplication Events in the Brassicales

Whole-genome duplications (WGDs) are prevalent throughout the evolutionary history of plants. For example, dozens of WGDs have been phylogenetically localized across the order Brassicales, specifically, within the family Brassicaceae. However, while its sister family, Cleomaceae, has also been characterized by a WGD, its placement, as well as that of other WGD events in other families in the order, remains unclear. Using phylo-transcriptomics from 74 taxa and genome survey sequencing for 66 of those taxa, we infer nuclear and chloroplast phylogenies to assess relationships among the major families of the Brassicales and within the Brassicaceae. We then use multiple methods of WGD inference to assess placement of WGD events. We not only present well-supported chloroplast and nuclear phylogenies for the Brassicales, but we also putatively place Th- and provide evidence for previously unknown events, including one shared by at least two members of the Resedaceae, which we name Rs-. Given its economic importance and many genomic resources, the Brassicales are an ideal group to continue assessing WGD inference methods. We add to the current conversation on WGD inference difficulties, by demonstrating that sampling is especially important for WGD identification.

evolutionary biology↗

bioRxiv2019

A blueprint of seed desiccation sensitivity in the genome of Castanospermum australe

O_LIMost angiosperms produce seeds that are desiccated on dispersal with the ability to retain viability in storage facilities for prolonged periods. However, some species produce desiccation sensitive seeds which rapidly lose viability in storage, precluding ex situ conservation. Current consensus is that desiccation sensitive seeds either lack or do not express mechanisms necessary for the acquisition of desiccation tolerance.\nC_LIO_LIWe sequenced the genome of Castanospermum australe, a legume species producing desiccation sensitive seeds, and characterized its seed developmental physiology and - transcriptomes.\nC_LIO_LIC. australe has a low rate of evolution, likely due to its perennial life-cycle and long generation times. The genome is syntenic with itself, with several orthologs of genes from desiccation tolerant legume seeds, from gamma whole-genome duplication events being retained. Changes in gene expression during development of C. australe seeds, as compared to desiccation tolerant Medicago truncatula seeds, suggest they remain metabolically active, prepared for immediate germination.\nC_LIO_LIOur data indicates that the phenotype of C. australe seeds arose through few changes in specific signalling pathways, precluding or bypassing activation of mechanisms necessary for acquisition of desiccation tolerance. Such changes have been perpetuated as the habitat in which dispersal occurs is favourable for prompt germination.\nC_LI

plant biology↗

bioRxiv2019

Genome improvement and genetic map construction for Aethionema arabicum, the first divergent branch in the Brassicaceae family

BackgroundThe genus Aethionema is a sister-group to the core-group of the Brassicaceae family that includes Arabidopsis thaliana and the Brassica crops. Thus, Aethionema is phylogenetically well-placed for the investigation and understanding of genome and trait evolution across the family. We aimed to improve the quality of the reference genome draft version of the annual species Aethionema arabicum. Secondly, we constructed the first Ae. arabicum genetic map. The improved reference genome and genetic map enabled the development of each other.\n\nResultsWe started with the initially published genome (version 2.5). PacBio and MinION sequencing together with genetic map v2.5 were incorporated to produce the new reference genome v3.0. The improved genome contains 203 MB of sequence, with approximately 94% of the assembly made up of called bases, assembled into 2,883 scaffolds. The N50 (10.3 MB) represents an 80-fold over the initial genome release. We generated a Recombinant Inbred Line (RIL) population that was derived from two ecotypes: Cyprus and Turkey (the reference genotype. Using a Genotyping by Sequencing (GBS) approach, we generated a high-density genetic map with 749 (v2.5) and then 632 SNPs (v3.0) was generated. The genetic map and reference genome were integrated, thus greatly improving the scaffolding of the reference genome into 11 linkage groups.\n\nConclusionsWe show that long-read sequencing data and genetics are complementary, resulting in an improved genome assembly in Ae. arabicum. They will facilitate comparative genetic mapping work for the Brassicaceae family and are also valuable resources to investigate wide range of life history traits in Aethionema.

genomics↗