The Jan Sjödin faba bean mutant collection: morphological and molecular characterization.

BACKGROUND: Plant mutagenesis creates novel alleles, thereby increasing genetic and phenotypic diversity. The availability of the faba bean (Vicia faba L.) reference genome and a growing set of additional genomic resources has increased the scientific and practical value of mutant collections. We aimed to genotype and morphologically phenotype a historical faba bean mutant collection developed and characterized by Jan Sjödin (1934-2023) over half a century ago in order to increase its value to researchers. The collection was genotyped using high-throughput single-primer enrichment technology (SPET) assays. RESULTS: We used 11,073 informative single nucleotide polymorphism (SNP) markers spanning the faba bean genome to genotype 52 mutant lines along with the background line, cv. Primus. A range of flower, seed, leaf, and stipule mutations were observed. The analysis of population structure revealed a shallow structure with no major subpopulations. Principal component and cluster analyses revealed, to a minor extent, that the mutants clustered by their phenotype. CONCLUSIONS: The mutants' phenotypic variation and shallow structure indicate that the Sjödin faba bean collection has the potential to play a significant role in faba bean breeding and in genetic and functional studies.

Multi-model GWAS reveals key loci for horticultural traits in reconstructed garden strawberry.

The cultivated garden strawberry (Fragaria × ananassa) has a rich history, originating from the hybridization of two wild octoploid strawberry species in the 18th century. Two-step reconstruction of Fragaria × ananassa through controlled crossings between pre-improved selections of its parental species is a promising approach for enriching the breeding germplasm of strawberry for wider adaptability. We created a population of reconstructed strawberry by hybridizing elite selections of F. virginiana and F. chiloensis. A replicated field experiment was conducted to evaluate the population's performance for eleven horticulturally important traits, over multiple years. Population structure analyses based on Fana-50 k SNP array data confirmed pedigree-based grouping of the progenies into four distinct groups. As complex traits are often influenced by environmental variables, and population structure can lead to spurious associations, we tested multiple genome-wide association study (GWAS) models. GWAS uncovered 39 quantitative trait loci (QTL) regions for eight traits distributed across twenty chromosomes, including 11 consistent and 28 putative QTLs. Candidate genes for traits including winter survival, flowering time, runnering vigor, and hermaphrodism were identified within the QTL regions. To our knowledge, this study marks the first comprehensive investigation of adaptive and horticultural traits in a large, multi-familial reconstructed strawberry population using SNP markers.

Precision phenotyping of a barley diversity set reveals distinct drought response strategies.

Plants exhibit an array of drought responses and adaptations, where the trade-off between water loss and CO2 uptake for growth is mediated by regulation of stomatal aperture in response to soil water content (SWC), among other factors. For crop yield stability, the question is how drought timing and response patterns relate to post-drought growth resilience and vigor. We earlier identified, in a few reference varieties of barley that differed by the SWC at which transpiration was curtailed, two divergent water use strategies: water-saving ("isohydric") and water-spending ("anisohydric"). We proposed that an isohydric strategy may reduce risk from spring droughts in climates where the probability of precipitation increases during the growing season, whereas the anisohydric is consistent with environments having terminal droughts, or with those where dry periods are short and not seasonally progressive. Here, we have examined drought response physiology in an 81-line barley (Hordeum vulgare L.) diversity set that spans 20th century European breeding and identified several lines with a third, dynamic strategy. We found a strong positive correlation between vigor and transpiration, the dynamic group being highest for both. However, these lines curtailed daily transpiration at a higher SWC than the isohydric group. While the dynamic lines, particularly cv Hydrogen and Baronesse, were not the most resilient in terms of restoring initial growth rates, their strong initial vigor and high return to initial transpiration rates meant that their growth nevertheless surpassed more resilient lines during recovery from drought. The results will be of use for defining barley physiological ideotypes suited to future climate scenarios.

Drought response of water-conserving and non-conserving spring barley cultivars.

Introduction: Breeding barley cultivars adapted to drought requires in-depth knowledge on physiological drought responses. Methods: We used a high-throughput functional phenotyping platform to examine the response of four high-yielding European spring barley cultivars to a standardized drought treatment imposed around flowering. Results: Cv. Chanell showed a non-conserving water-use behavior with high transpiration and maximum productivity under well-watered conditions but rapid transpiration decrease under drought. The poor recovery upon re-irrigation translated to large yield losses. Cv. Baronesse showed the most water-conserving behavior, with the lowest pre-drought transpiration and the most gradual transpiration reduction under drought. Its good recovery (resilience) prevented large yield losses. Cv. Formula was less conserving than cv. Baronesse and produced low yet stable yields. Cv. RGT's dynamic water use with high transpiration under ample water supply and moderate transpiration decrease under drought combined with high resilience secured the highest and most stable yields. Discussion: Such a dynamic water-use behavior combined with higher drought resilience and favorable root traits could potentially create an ideotype for intermediate drought. Prospective studies will examine these results in field experiments and will use the newly gained understanding on water use in barley to improve process descriptions in crop simulation models to support crop model-aided ideotype design.

Drought and recovery in barley: key gene networks and retrotransposon response.

Introduction: During drought, plants close their stomata at a critical soil water content (SWC), together with making diverse physiological, developmental, and biochemical responses. Methods: Using precision-phenotyping lysimeters, we imposed pre-flowering drought on four barley varieties (Arvo, Golden Promise, Hankkija 673, and Morex) and followed their physiological responses. For Golden Promise, we carried out RNA-seq on leaf transcripts before and during drought and during recovery, also examining retrotransposon BARE1expression. Transcriptional data were subjected to network analysis. Results: The varieties differed by their critical SWC (Ï´crit), Hankkija 673 responding at the highest and Golden Promise at the lowest. Pathways connected to drought and salinity response were strongly upregulated during drought; pathways connected to growth and development were strongly downregulated. During recovery, growth and development pathways were upregulated; altogether, 117 networked genes involved in ubiquitin-mediated autophagy were downregulated. Discussion: The differential response to SWC suggests adaptation to distinct rainfall patterns. We identified several strongly differentially expressed genes not earlier associated with drought response in barley. BARE1 transcription is strongly transcriptionally upregulated by drought and downregulated during recovery unequally between the investigated cultivars. The downregulation of networked autophagy genes suggests a role for autophagy in drought response; its importance to resilience should be further investigated.

Understanding and applying biological resilience, from genes to ecosystems.

The natural world is under unprecedented and accelerating pressure. Much work on understanding resilience to local and global environmental change has, so far, focussed on ecosystems. However, understanding a system's behaviour requires knowledge of its component parts and their interactions. Here we call for increased efforts to understand 'biological resilience', or the processes that enable components across biological levels, from genes to communities, to resist or recover from perturbations. Although ecologists and evolutionary biologists have the tool-boxes to examine form and function, efforts to integrate this knowledge across biological levels and take advantage of big data (e.g. ecological and genomic) are only just beginning. We argue that combining eco-evolutionary knowledge with ecosystem-level concepts of resilience will provide the mechanistic basis necessary to improve management of human, natural and agricultural ecosystems, and outline some of the challenges in achieving an understanding of biological resilience.

Multi-environment genome -wide association mapping of culm morphology traits in barley.

In cereals with hollow internodes, lodging resistance is influenced by morphological characteristics such as internode diameter and culm wall thickness. Despite their relevance, knowledge of the genetic control of these traits and their relationship with lodging is lacking in temperate cereals such as barley. To fill this gap, we developed an image analysis-based protocol to accurately phenotype culm diameters and culm wall thickness across 261 barley accessions. Analysis of culm trait data collected from field trials in seven different environments revealed high heritability values (>50%) for most traits except thickness and stiffness, as well as genotype-by-environment interactions. The collection was structured mainly according to row-type, which had a confounding effect on culm traits as evidenced by phenotypic correlations. Within both row-type subsets, outer diameter and section modulus showed significant negative correlations with lodging (<-0.52 and <-0.45, respectively), but no correlation with plant height, indicating the possibility of improving lodging resistance independent of plant height. Using 50k iSelect SNP genotyping data, we conducted multi-environment genome-wide association studies using mixed model approach across the whole panel and row-type subsets: we identified a total of 192 quantitative trait loci (QTLs) for the studied traits, including subpopulation-specific QTLs and 21 main effect loci for culm diameter and/or section modulus showing effects on lodging without impacting plant height. Providing insights into the genetic architecture of culm morphology in barley and the possible role of candidate genes involved in hormone and cell wall-related pathways, this work supports the potential of loci underpinning culm features to improve lodging resistance and increase barley yield stability under changing environments.

Genomic regions associated with chocolate spot (Botrytis fabae Sard.) resistance in faba bean (Vicia faba L.).

Chocolate spot (CS), caused by Botrytis fabae Sard., is an important threat to global faba bean production. Growing resistant faba bean cultivars is, therefore, paramount to preventing yield loss. To date, there have been no reported quantitative trait loci (QTL) associated with CS resistance in faba bean. The objective of this study was to identify genomic regions associated with CS resistance using a recombinant inbred line (RIL) population derived from resistant accession ILB 938. A total of 165 RILs from the cross Mélodie/2 × ILB 938/2 were genotyped and evaluated for CS reactions under replicated controlled climate conditions. The RIL population showed significant variation in response to CS resistance. QTL analysis identified five loci contributing to CS resistance on faba bean chromosomes 1 and 6, accounting for 28.4% and 12.5%, respectively, of the total phenotypic variance. The results of this study not only provide insight into disease-resistance QTL, but also can be used as potential targets for marker-assisted breeding in faba bean genetic improvement for CS resistance. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01307-7.

Palindromic Sequence-Targeted (PST) PCR, Version 2: An Advanced Method for High-Throughput Targeted Gene Characterization and Transposon Display.

Genome walking (GW), a strategy for capturing previously unsequenced DNA fragments that are in proximity to a known sequence tag, is currently predominantly based on PCR. Recently developed PCR-based methods allow for combining of sequence-specific primers with designed capturing primers capable of annealing to unknown DNA targets, thereby offering the rapidity and effectiveness of PCR. This study presents a methodological improvement to the previously described GW technique known as palindromic sequence-targeted PCR (PST-PCR). Like PST-PCR, this new method (called PST-PCR v.2) relies on targeting of capturing primers to palindromic sequences arbitrarily present in natural DNA templates. PST-PCR v.2 consists of two rounds of PCR. The first round uses a combination of one sequence-specific primer with one capturing (PST) primer. The second round uses a combination of a single (preferred) or two universal primers; one anneals to a 5' tail attached to the sequence-specific primer and the other anneals to a different 5' tail attached to the PST primer. The key advantage of PST-PCR v.2 is the convenience of using a single universal primer with invariable sequences in GW processes involving various templates. The entire procedure takes approximately 2-3 h to produce the amplified PCR fragment, which contains a portion of a template flanked by the sequence-specific and capturing primers. PST-PCR v.2 is highly suitable for simultaneous work with multiple samples. For this reason, PST-PCR v.2 can be applied beyond the classical task of GW for studies in population genetics, in which PST-PCR v.2 is a preferred alternative to amplified fragment length polymorphism (AFLP) or next-generation sequencing. Furthermore, the conditions for PST-PCR v.2 are easier to optimize, as only one sequence-specific primer is used. This reduces non-specific random amplified polymorphic DNA (RAPD)-like amplification and formation of non-templated amplification. Importantly, akin to the previous version, PST-PCR v.2 is not sensitive to template DNA sequence complexity or quality. This study illustrates the utility of PST-PCR v.2 for transposon display (TD), which is a method to characterize inter- or intra-specific variability related to transposon integration sites. The Ac transposon sequence in the maize (Zea mays) genome was used as a sequence tag during the TD procedure to characterize the Ac integration sites.

VC1 catalyses a key step in the biosynthesis of vicine in faba bean.

Faba bean (Vicia faba L.) is a widely adapted and high-yielding legume cultivated for its protein-rich seeds1. However, the seeds accumulate the pyrimidine glucosides vicine and convicine, which can cause haemolytic anaemia (favism) in 400 million genetically predisposed individuals2. Here, we use gene-to-metabolite correlations, gene mapping and genetic complementation to identify VC1 as a key enzyme in vicine and convicine biosynthesis. We demonstrate that VC1 has GTP cyclohydrolase II activity and that the purine GTP is a precursor of both vicine and convicine. Finally, we show that cultivars with low vicine and convicine levels carry an inactivating insertion in the coding sequence of VC1. Our results reveal an unexpected, purine rather than pyrimidine, biosynthetic origin for vicine and convicine and pave the way for the development of faba bean cultivars that are free of these anti-nutrients.

Transposon-Based Tagging In Silico Using FastPCR Software.

Retrotransposons are ubiquitous, generally dispersed components of eukaryotic genomes. These properties, together with their "copy and paste" lifecycle that generates insertional polymorphism without need for excision, makes them widely useful as a molecular-genetic tags. Various tagging systems have been developed that exploit the sequence conservation of retrotransposon components, such as those found in their long terminal repeats (LTRs). To detect polymorphisms for retrotransposon insertions, marker systems generally rely on PCR amplification between the termini and some component of flanking genomic DNA. As complements to various "wet lab" protocols for retrotransposon tagging, in silico bioinformatics approaches are useful for predicting likely outcomes from unsequenced accessions on the basis of reference genomes. In this chapter, we describe protocols for in silico retrotransposon-based fingerprinting techniques using the FastPCR software as an integrated tools environment for in silico PCR primer design and analysis.

Recent advances in faba bean genetic and genomic tools for crop improvement.

Faba bean (Vicia faba L.), a member of the Fabaceae family, is one of the important food legumes cultivated in cool temperate regions. It holds great importance for human consumption and livestock feed because of its high protein content, dietary fibre, and nutritional value. Major faba bean breeding challenges include its mixed breeding system, unknown wild progenitor, and genome size of ~13 Gb, which is the largest among diploid field crops. The key breeding objectives in faba bean include improved resistance to biotic and abiotic stress and enhanced seed quality traits. Regarding quality traits, major progress on reduction of vicine-convicine and seed coat tannins, the main anti-nutritional factors limiting faba bean seed usage, have been recently achieved through gene discovery. Genomic resources are relatively less advanced compared with other grain legume species, but significant improvements are underway due to a recent increase in research activities. A number of bi-parental populations have been constructed and mapped for targeted traits in the last decade. Faba bean now benefits from saturated synteny-based genetic maps, along with next-generation sequencing and high-throughput genotyping technologies that are paving the way for marker-assisted selection. Developing a reference genome, and ultimately a pan-genome, will provide a foundational resource for molecular breeding. In this review, we cover the recent development and deployment of genomic tools for faba bean breeding.

Long Tandem Arrays of Cassandra Retroelements and Their Role in Genome Dynamics in Plants.

Retrotransposable elements are widely distributed and diverse in eukaryotes. Their copy number increases through reverse-transcription-mediated propagation, while they can be lost through recombinational processes, generating genomic rearrangements. We previously identified extensive structurally uniform retrotransposon groups in which no member contains the gag, pol, or env internal domains. Because of the lack of protein-coding capacity, these groups are non-autonomous in replication, even if transcriptionally active. The Cassandra element belongs to the non-autonomous group called terminal-repeat retrotransposons in miniature (TRIM). It carries 5S RNA sequences with conserved RNA polymerase (pol) III promoters and terminators in its long terminal repeats (LTRs). Here, we identified multiple extended tandem arrays of Cassandra retrotransposons within different plant species, including ferns. At least 12 copies of repeated LTRs (as the tandem unit) and internal domain (as a spacer), giving a pattern that resembles the cellular 5S rRNA genes, were identified. A cytogenetic analysis revealed the specific chromosomal pattern of the Cassandra retrotransposon with prominent clustering at and around 5S rDNA loci. The secondary structure of the Cassandra retroelement RNA is predicted to form super-loops, in which the two LTRs are complementary to each other and can initiate local recombination, leading to the tandem arrays of Cassandra elements. The array structures are conserved for Cassandra retroelements of different species. We speculate that recombination events similar to those of 5S rRNA genes may explain the wide variation in Cassandra copy number. Likewise, the organization of 5S rRNA gene sequences is very variable in flowering plants; part of what is taken for 5S gene copy variation may be variation in Cassandra number. The role of the Cassandra 5S sequences remains to be established.

High-throughput retrotransposon-based genetic diversity of maize germplasm assessment and analysis.

Maize is one of the world's most important crops and a model for grass genome research. Long terminal repeat (LTR) retrotransposons comprise most of the maize genome; their ability to produce new copies makes them efficient high-throughput genetic markers. Inter-retrotransposon-amplified polymorphisms (IRAPs) were used to study the genetic diversity of maize germplasm. Five LTR retrotransposons (Huck, Tekay, Opie, Ji, and Grande) were chosen, based on their large number of copies in the maize genome, whereas polymerase chain reaction primers were designed based on consensus LTR sequences. The LTR primers showed high quality and reproducible DNA fingerprints, with a total of 677 bands including 392 polymorphic bands showing 58% polymorphism between maize hybrid lines. These markers were used to identify genetic similarities among all lines of maize. Analysis of genetic similarity was carried out based on polymorphic amplicon profiles and genetic similarity phylogeny analysis. This diversity was expected to display ecogeographical patterns of variation and local adaptation. The clustering method showed that the varieties were grouped into three clusters differing in ecogeographical origin. Each of these clusters comprised divergent hybrids with convergent characters. The clusters reflected the differences among maize hybrids and were in accordance with their pedigree. The IRAP technique is an efficient high-throughput genetic marker-generating method.

TRITEX: chromosome-scale sequence assembly of Triticeae genomes with open-source tools.

Chromosome-scale genome sequence assemblies underpin pan-genomic studies. Recent genome assembly efforts in the large-genome Triticeae crops wheat and barley have relied on the commercial closed-source assembly algorithm DeNovoMagic. We present TRITEX, an open-source computational workflow that combines paired-end, mate-pair, 10X Genomics linked-read with chromosome conformation capture sequencing data to construct sequence scaffolds with megabase-scale contiguity ordered into chromosomal pseudomolecules. We evaluate the performance of TRITEX on publicly available sequence data of tetraploid wild emmer and hexaploid bread wheat, and construct an improved annotated reference genome sequence assembly of the barley cultivar Morex as a community resource.

Palindromic sequence-targeted (PST) PCR: a rapid and efficient method for high-throughput gene characterization and genome walking.

Genome walking (GW) refers to the capture and sequencing of unknown regions in a long DNA molecule that are adjacent to a region with a known sequence. A novel PCR-based method, palindromic sequence-targeted PCR (PST-PCR), was developed. PST-PCR is based on a distinctive design of walking primers and special thermal cycling conditions. The walking primers (PST primers) match palindromic sequences (PST sites) that are randomly distributed in natural DNA. The PST primers have palindromic sequences at their 3'-ends. Upstream of the palindromes there is a degenerate sequence (8-12 nucleotides long); defined adapters are present at the 5'-termini. The thermal cycling profile has a linear amplification phase and an exponential amplification phase differing in annealing temperature. Changing the annealing temperature to switch the amplification phases at a defined cycle controls the balance between sensitivity and specificity. In contrast to traditional genome walking methods, PST-PCR is rapid (two to three hours to produce GW fragments) as it uses only one or two PCR rounds. Using PST-PCR, previously unknown regions (the promoter and intron 1) of the VRN1 gene of Timothy-grass (Phleum pratense L.) were captured for sequencing. In our experience, PST-PCR had higher throughput and greater convenience in comparison to other GW methods.

Genome Sequence of Striga asiatica Provides Insight into the Evolution of Plant Parasitism.

Parasitic plants in the genus Striga, commonly known as witchweeds, cause major crop losses in sub-Saharan Africa and pose a threat to agriculture worldwide. An understanding of Striga parasite biology, which could lead to agricultural solutions, has been hampered by the lack of genome information. Here, we report the draft genome sequence of Striga asiatica with 34,577 predicted protein-coding genes, which reflects gene family contractions and expansions that are consistent with a three-phase model of parasitic plant genome evolution. Striga seeds germinate in response to host-derived strigolactones (SLs) and then develop a specialized penetration structure, the haustorium, to invade the host root. A family of SL receptors has undergone a striking expansion, suggesting a molecular basis for the evolution of broad host range among Striga spp. We found that genes involved in lateral root development in non-parasitic model species are coordinately induced during haustorium development in Striga, suggesting a pathway that was partly co-opted during the evolution of the haustorium. In addition, we found evidence for horizontal transfer of host genes as well as retrotransposons, indicating gene flow to S. asiatica from hosts. Our results provide valuable insights into the evolution of parasitism and a key resource for the future development of Striga control strategies.

The genome of cowpea (Vigna unguiculata [L.] Walp.).

Cowpea (Vigna unguiculata [L.] Walp.) is a major crop for worldwide food and nutritional security, especially in sub-Saharan Africa, that is resilient to hot and drought-prone environments. An assembly of the single-haplotype inbred genome of cowpea IT97K-499-35 was developed by exploiting the synergies between single-molecule real-time sequencing, optical and genetic mapping, and an assembly reconciliation algorithm. A total of 519 Mb is included in the assembled sequences. Nearly half of the assembled sequence is composed of repetitive elements, which are enriched within recombination-poor pericentromeric regions. A comparative analysis of these elements suggests that genome size differences between Vigna species are mainly attributable to changes in the amount of Gypsy retrotransposons. Conversely, genes are more abundant in more distal, high-recombination regions of the chromosomes; there appears to be more duplication of genes within the NBS-LRR and the SAUR-like auxin superfamilies compared with other warm-season legumes that have been sequenced. A surprising outcome is the identification of an inversion of 4.2 Mb among landraces and cultivars, which includes a gene that has been associated in other plants with interactions with the parasitic weed Striga gesnerioides. The genome sequence facilitated the identification of a putative syntelog for multiple organ gigantism in legumes. A revised numbering system has been adopted for cowpea chromosomes based on synteny with common bean (Phaseolus vulgaris). An estimate of nuclear genome size of 640.6 Mbp based on cytometry is presented.

Microsatellite Diversity, Complexity, and Host Range of Mycobacteriophage Genomes of the Siphoviridae Family.

The incidence, distribution, and variation of simple sequence repeats (SSRs) in viruses is instrumental in understanding the functional and evolutionary aspects of repeat sequences. Full-length genome sequences retrieved from NCBI were used for extraction and analysis of repeat sequences using IMEx software. We have also developed two MATLAB-based tools for extraction of gene locations from GenBank in tabular format and simulation of this data with SSR incidence data. Present study encompassing 147 Mycobacteriophage genomes revealed 25,284 SSRs and 1,127 compound SSRs (cSSRs) through IMEx. Mono- to hexa-nucleotide motifs were present. The SSR count per genome ranged from 78 (M100) to 342 (M58) while cSSRs incidence ranged from 1 (M138) to 17 (M28, M73). Though cSSRs were present in all the genomes, their frequency and SSR to cSSR conversion percentage varied from 1.08 (M138 with 93 SSRs) to 8.33 (M116 with 96 SSRs). In terms of localization, the SSRs were predominantly localized to coding regions (∼78%). Interestingly, genomes of around 50 kb contained a similar number of SSRs/cSSRs to that in a 110 kb genome, suggesting functional relevance for SSRs which was substantiated by variation in motif constitution between species with different host range. The three species with broad host range (M97, M100, M116) have around 90% of their mono-nucleotide repeat motifs composed of G or C and only M16 has both A and T mononucleotide motifs. Around 20% of the di-nucleotide repeat motifs in the genomes exhibiting a broad host range were CT/TC, which were either absent or represented to a much lesser extent in the other genomes.