Genetic diversity and candidate genes for transient waterlogging tolerance in mungbean at the germination and seedling stages.

Mungbean [Vigna radiata var. radiata (L.) Wilczek] production in Asia is detrimentally affected by transient soil waterlogging caused by unseasonal and increasingly frequent extreme precipitation events. While mungbean exhibits sensitivity to waterlogging, there has been insufficient exploration of germplasm for waterlogging tolerance, as well as limited investigation into the genetic basis for tolerance to identify valuable loci. This research investigated the diversity of transient waterlogging tolerance in a mini-core germplasm collection of mungbean and identified candidate genes for adaptive traits of interest using genome-wide association studies (GWAS) at two critical stages of growth: germination and seedling stage (i.e., once the first trifoliate leaf had fully-expanded). In a temperature-controlled glasshouse, 292 genotypes were screened for tolerance after (i) 4 days of waterlogging followed by 7 days of recovery at the germination stage and (ii) 8 days of waterlogging followed by 7 days of recovery at the seedling stage. Tolerance was measured against drained controls. GWAS was conducted using 3,522 high-quality DArTseq-derived SNPs, revealing five significant associations with five phenotypic traits indicating improved tolerance. Waterlogging tolerance was positively correlated with the formation of adventitious roots and higher dry masses. FGGY carbohydrate kinase domain-containing protein was identified as a candidate gene for adventitious rooting and mRNA-uncharacterized LOC111241851, Caffeoyl-CoA O-methyltransferase At4g26220 and MORC family CW-type zinc finger protein 3 and zinc finger protein 2B genes for shoot, root, and total dry matter production. Moderate to high broad-sense heritability was exhibited for all phenotypic traits, including seed emergence (81%), adventitious rooting (56%), shoot dry mass (81%), root dry mass (79%) and SPAD chlorophyll content (70%). The heritability estimates, marker-trait associations, and identification of sources of waterlogging tolerant germplasm from this study demonstrate high potential for marker-assisted selection of tolerance traits to accelerate breeding of climate-resilient mungbean varieties.

Distribution, frequency and molecular basis of clethodim and quizalofop resistance in brome grass (Bromus diandrus).

BACKGROUND: Brome grass (Bromus diandrus Roth) is prevalent in the southern and western cropping regions of Australia, where it causes significant economic damage. A targeted herbicide resistance survey was conducted in 2020 by collecting brome grass populations from 40 farms in Western Australia and subjecting these samples to comprehensive herbicide screening. One sample (population 172-20), from a field that had received 12 applications of clethodim over 20 years of continuous cropping, was found to be highly resistant to the acetyl-CoA carboxylase (ACCase)-inhibiting herbicides clethodim and quizalofop, and so the molecular basis of resistance was investigated. RESULTS: All 31 individuals examined from population 172-20 carried the same resistance-endowing point mutation causing an aspartate-to-glycine substitution at position 2078 in the translated ACCase protein sequence. A wild-type susceptible population and the resistant population had similar expression levels of plastidic ACCase genes. The level of resistance to quizalofop, either standalone or in mixture with clethodim, in population 172-20 was lower under cooler growing conditions. CONCLUSION: Target-site resistance to ACCase-inhibiting herbicides, conferred by one ACCase mutation, was selected in all tested brome plants infesting a field with a history of repeated clethodim use. This mutation appears to have been fixed in the infesting population. Notably, clethodim resistance in this population was not detected by the farmer, and a high future incidence of quizalofop resistance is anticipated. Herbicide resistance testing is essential for the detection of evolving weed resistance issues and to inform effective management strategies. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Gain or Loss? Evidence for Legume Predisposition to Symbiotic Interactions with Rhizobia via Loss of Pathogen-Resistance-Related Gene Families.

Nodulation is a hallmark yet non-universal characteristic of legumes. It is unknown whether the mechanisms underlying nitrogen-fixing symbioses evolved within legumes and the broader nitrogen-fixing clade (NFC) repeatedly de novo or based on common ancestral pathways. Ten new transcriptomes representing members from the Cercidoideae and Caesalpinioideae subfamilies were supplemented with published omics data from 65 angiosperms, to investigate how gene content correlates with nodulation capacity within Fabaceae and the NFC. Orthogroup analysis categorized annotated genes into 64150 orthogroups, of which 19 were significantly differentially represented between nodulating versus non-nodulating NFC species and were most commonly absent in nodulating taxa. The distribution of six over-represented orthogroups within Viridiplantae representatives suggested that genomic evolution events causing gene family expansions, including whole-genome duplications (WGDs), were unlikely to have facilitated the development of stable symbioses within Fabaceae as a whole. Instead, an absence of representation of 13 orthogroups indicated that losses of genes involved in trichome development, defense and wounding responses were strongly associated with rhizobial symbiosis in legumes. This finding provides novel evidence of a lineage-specific predisposition for the evolution and/or stabilization of nodulation in Fabaceae, in which a loss of pathogen resistance genes may have allowed for stable mutualistic interactions with rhizobia.

A pan-genome and chromosome-length reference genome of narrow-leafed lupin (Lupinus angustifolius) reveals genomic diversity and insights into key industry and biological traits.

Narrow-leafed lupin (NLL; Lupinus angustifolius) is a key rotational crop for sustainable farming systems, whose grain is high in protein content. It is a gluten-free, non-genetically modified, alternative protein source to soybean (Glycine max) and as such has gained interest as a human food ingredient. Here, we present a chromosome-length reference genome for the species and a pan-genome assembly comprising 55 NLL lines, including Australian and European cultivars, breeding lines and wild accessions. We present the core and variable genes for the species and report on the absence of essential mycorrhizal associated genes. The genome and pan-genomes of NLL and its close relative white lupin (Lupinus albus) are compared. Furthermore, we provide additional evidence supporting LaRAP2-7 as the key alkaloid regulatory gene for NLL and demonstrate the NLL genome is underrepresented in classical NLR disease resistance genes compared to other sequenced legume species. The NLL genomic resources generated here coupled with previously generated RNA sequencing datasets provide new opportunities to fast-track lupin crop improvement.

A Trimethylguanosine Synthase1-like (TGS1) homologue is implicated in vernalisation and flowering time control.

KEY MESSAGE: A plant-specific Trimethylguanosine Synthase1-like homologue was identified as a candidate gene for the efl mutation in narrow-leafed lupin, which alters phenology by reducing vernalisation requirement. The vernalisation pathway is a key component of flowering time control in plants from temperate regions but is not well understood in the legume family. Here we examined vernalisation control in the temperate grain legume species, narrow-leafed lupin (Lupinus angustifolius L.), and discovered a candidate gene for an ethylene imine mutation (efl). The efl mutation changes phenology from late to mid-season flowering and additionally causes transformation from obligate to facultative vernalisation requirement. The efl locus was mapped to pseudochromosome NLL-10 in a recombinant inbred line (RIL) mapping population developed by accelerated single seed descent. Candidate genes were identified in the reference genome, and a diverse panel of narrow-leafed lupins was screened to validate mutations specific to accessions with efl. A non-synonymous SNP mutation within an S-adenosyl-L-methionine-dependent methyltransferase protein domain of a Trimethylguanosine Synthase1-like (TGS1) orthologue was identified as the candidate mutation giving rise to efl. This mutation caused substitution of an amino acid within an established motif at a position that is otherwise highly conserved in several plant families and was perfectly correlated with the efl phenotype in F2 and F6 genetic population and a panel of diverse accessions, including the original efl mutant. Expression of the TGS1 homologue did not differ between wild-type and efl genotypes, supporting altered functional activity of the gene product. This is the first time a TGS1 orthologue has been associated with vernalisation response and flowering time control in any plant species.

INDEL variation in the regulatory region of the major flowering time gene LanFTc1 is associated with vernalization response and flowering time in narrow-leafed lupin (Lupinus angustifolius L.).

Narrow-leafed lupin (Lupinus angustifolius L.) cultivation was transformed by 2 dominant vernalization-insensitive, early flowering time loci known as Ku and Julius (Jul), which allowed expansion into shorter season environments. However, reliance on these loci has limited genetic and phenotypic diversity for environmental adaptation in cultivated lupin. We recently predicted that a 1,423-bp deletion in the cis-regulatory region of LanFTc1, a FLOWERING LOCUS T (FT) homologue, derepressed expression of LanFTc1 and was the underlying cause of the Ku phenotype. Here, we surveyed diverse germplasm for LanFTc1 cis-regulatory variation and identified 2 further deletions of 1,208 and 5,162 bp in the 5' regulatory region, which overlap the 1,423-bp deletion. Additionally, we confirmed that no other polymorphisms were perfectly associated with vernalization responsiveness. Phenotyping and gene expression analyses revealed that Jul accessions possessed the 5,162-bp deletion and that the Jul and Ku deletions were equally capable of removing vernalization requirement and up-regulating gene expression. The 1,208-bp deletion was associated with intermediate phenology, vernalization responsiveness, and gene expression and therefore may be useful for expanding agronomic adaptation of lupin. This insertion/deletion series may also help resolve how the vernalization response is mediated at the molecular level in legumes.

The loss of vernalization requirement in narrow-leafed lupin is associated with a deletion in the promoter and de-repressed expression of a Flowering Locus T (FT) homologue.

Adaptation of Lupinus angustifolius (narrow-leafed lupin) to cropping in southern Australian and northern Europe was transformed by a dominant mutation (Ku) that removed vernalization requirement for flowering. The Ku mutation is now widely used in lupin breeding to confer early flowering and maturity. We report here the identity of the Ku mutation. We used a range of genetic, genomic and gene expression approaches to determine whether Flowering Locus T (FT) homologues are associated with the Ku locus. One of four FT homologues present in the narrow-leafed lupin genome, LanFTc1, perfectly co-segregated with the Ku locus in a reference mapping population. Expression of LanFTc1 in the ku (late-flowering) parent was strongly induced by vernalization, in contrast to the Ku (early-flowering) parent, which showed constitutively high LanFTc1 expression. Co-segregation of this expression phenotype with the LanFTc1 genotype indicated that the Ku mutation impairs cis-regulation of LanFTc1. Sequencing of LanFTc1 revealed a 1.4-kb deletion in the promoter region, which was perfectly predictive of vernalization response in 216 wild and domesticated accessions. Linkage disequilibrium rapidly decayed around LanFTc1, suggesting that this deletion caused the loss of vernalization response. This is the first time a legume FTc subclade gene has been implicated in the vernalization response.

Identifying Stable Reference Genes for qRT-PCR Normalisation in Gene Expression Studies of Narrow-Leafed Lupin (Lupinus angustifolius L.).

Quantitative Reverse Transcription PCR (qRT-PCR) is currently one of the most popular, high-throughput and sensitive technologies available for quantifying gene expression. Its accurate application depends heavily upon normalisation of gene-of-interest data with reference genes that are uniformly expressed under experimental conditions. The aim of this study was to provide the first validation of reference genes for Lupinus angustifolius (narrow-leafed lupin, a significant grain legume crop) using a selection of seven genes previously trialed as reference genes for the model legume, Medicago truncatula. In a preliminary evaluation, the seven candidate reference genes were assessed on the basis of primer specificity for their respective targeted region, PCR amplification efficiency, and ability to discriminate between cDNA and gDNA. Following this assessment, expression of the three most promising candidates [Ubiquitin C (UBC), Helicase (HEL), and Polypyrimidine tract-binding protein (PTB)] was evaluated using the NormFinder and RefFinder statistical algorithms in two narrow-leafed lupin lines, both with and without vernalisation treatment, and across seven organ types (cotyledons, stem, leaves, shoot apical meristem, flowers, pods and roots) encompassing three developmental stages. UBC was consistently identified as the most stable candidate and has sufficiently uniform expression that it may be used as a sole reference gene under the experimental conditions tested here. However, as organ type and developmental stage were associated with greater variability in relative expression, it is recommended using UBC and HEL as a pair to achieve optimal normalisation. These results highlight the importance of rigorously assessing candidate reference genes for each species across a diverse range of organs and developmental stages. With emerging technologies, such as RNAseq, and the completion of valuable transcriptome data sets, it is possible that other potentially more suitable reference genes will be identified for this species in future.