Identification and expression analysis of Phosphate Transporter 1 (PHT1) genes in the highly phosphorus-use-efficient Hakea prostrata (Proteaceae).

Heavy and costly use of phosphorus (P) fertiliser is often needed to achieve high crop yields, but only a small amount of applied P fertiliser is available to most crop plants. Hakea prostrata (Proteaceae) is endemic to the P-impoverished landscape of southwest Australia and has several P-saving traits. We identified 16 members of the Phosphate Transporter 1 (PHT1) gene family (HpPHT1;1-HpPHT1;12d) in a long-read genome assembly of H. prostrata. Based on phylogenetics, sequence structure and expression patterns, we classified HpPHT1;1 as potentially involved in Pi uptake from soil and HpPHT1;8 and HpPHT1;9 as potentially involved in Pi uptake and root-to-shoot translocation. Three genes, HpPHT1;4, HpPHT1;6 and HpPHT1;8, lacked regulatory PHR1-binding sites (P1BS) in the promoter regions. Available expression data for HpPHT1;6 and HpPHT1;8 indicated they are not responsive to changes in P supply, potentially contributing to the high P sensitivity of H. prostrata. We also discovered a Proteaceae-specific clade of closely-spaced PHT1 genes that lacked conserved genetic architecture among genera, indicating an evolutionary hot spot within the genome. Overall, the genome assembly of H. prostrata provides a much-needed foundation for understanding the genetic mechanisms of novel adaptations to low P soils in southwest Australian plants.

Characterisation of common hypothetical surface peptides between protozoan parasites (Perkinsus olseni) originating from different geographical locations.

Perkinsus olseni and P. marinus are classified as notifiable pathogens by the World Organisation for Animal Health and are known to cause perkinsosis in a variety of molluscs globally. Mass mortalities due to these parasites in farms and in the wild have been a recurrent issue. Diagnosis for these protozoans is currently done using Ray's fluid thioglycollate medium method followed by optical microscopy or molecular assays. Both require a high level of skill and are time-consuming. An immunoassay method would make the diagnosis of perkinsosis quicker and cheaper. The present study used mass spectrometry-based proteomics to investigate common hypothetical surface peptides between different geographical isolates of P. olseni, which could be used to develop immunoassays in the future. Two peptides were identified: POLS_08089, which is a 42.7 kDa peptide corresponding to the 60S ribosomal subunit protein L4; and POLS_15916, which is a conserved hypothetical protein of 55.6 kDa. The identification of peptides may allow the development of immunoassays through a more targeted approach.

Extensive polyploid clonality was a successful strategy for seagrass to expand into a newly submerged environment.

Polyploidy has the potential to allow organisms to outcompete their diploid progenitor(s) and occupy new environments. Shark Bay, Western Australia, is a World Heritage Area dominated by temperate seagrass meadows including Poseidon's ribbon weed, Posidonia australis. This seagrass is at the northern extent of its natural geographic range and experiences extremes in temperature and salinity. Our genomic and cytogenetic assessments of 10 meadows identified geographically restricted, diploid clones (2n = 20) in a single location, and a single widespread, high-heterozygosity, polyploid clone (2n = 40) in all other locations. The polyploid clone spanned at least 180 km, making it the largest known example of a clone in any environment on earth. Whole-genome duplication through polyploidy, combined with clonality, may have provided the mechanism for P. australis to expand into new habitats and adapt to new environments that became increasingly stressful for its diploid progenitor(s). The new polyploid clone probably formed in shallow waters after the inundation of Shark Bay less than 8500 years ago and subsequently expanded via vegetative growth into newly submerged habitats.

Genotype-Environment mismatch of kelp forests under climate change.

Climate change is increasingly impacting ecosystems globally. Understanding adaptive genetic diversity and whether it will keep pace with projected climatic change is necessary to assess species' vulnerability and design efficient mitigation strategies such as assisted adaptation. Kelp forests are the foundations of temperate reefs globally but are declining in many regions due to climate stress. A lack of knowledge of kelp's adaptive genetic diversity hinders assessment of vulnerability under extant and future climates. Using 4245 single nucleotide polymorphisms (SNPs), we characterized patterns of neutral and putative adaptive genetic diversity for the dominant kelp in the southern hemisphere (Ecklonia radiata) from ~1000 km of coastline off Western Australia. Strong population structure and isolation-by-distance was underpinned by significant signatures of selection related to temperature and light. Gradient forest analysis of temperature-linked SNPs under selection revealed a strong association with mean annual temperature range, suggesting adaptation to local thermal environments. Critically, modelling revealed that predicted climate-mediated temperature changes will probably result in high genomic vulnerability via a mismatch between current and future predicted genotype-environment relationships such that kelp forests off Western Australia will need to significantly adapt to keep pace with projected climate change. Proactive management techniques such as assisted adaptation to boost resilience may be required to secure the future of these kelp forests and the immense ecological and economic values they support.

Resistance Gene Analogs in the Brassicaceae: Identification, Characterization, Distribution, and Evolution.

The Brassicaceae consists of a wide range of species, including important Brassica crop species and the model plant Arabidopsis (Arabidopsis thaliana). Brassica spp. crop diseases impose significant yield losses annually. A major way to reduce susceptibility to disease is the selection in breeding for resistance gene analogs (RGAs). Nucleotide binding site-leucine rich repeats (NLRs), receptor-like kinases (RLKs), and receptor-like proteins (RLPs) are the main types of RGAs; they contain conserved domains and motifs and play specific roles in resistance to pathogens. Here, all classes of RGAs have been identified using annotation and assembly-based pipelines in all available genome annotations from the Brassicaceae, including multiple genome assemblies of the same species where available (total of 32 genomes). The number of RGAs, based on genome annotations, varies within and between species. In total 34,065 RGAs were identified, with the majority being RLKs (21,691), then NLRs (8,588) and RLPs (3,786). Analysis of the RGA protein sequences revealed a high level of sequence identity, whereby 99.43% of RGAs fell into several orthogroups. This study establishes a resource for the identification and characterization of RGAs in the Brassicaceae and provides a framework for further studies of RGAs for an ultimate goal of assisting breeders in improving resistance to plant disease.

Candidate Rlm6 resistance genes against Leptosphaeria. maculans identified through a genome-wide association study in Brassica juncea (L.) Czern.

KEY MESSAGE: One hundred and sixty-seven B. juncea varieties were genotyped on the 90K Brassica assay (42,914 SNPs), which led to the identification of sixteen candidate genes for Rlm6. Brassica species are at high risk of severe crop loss due to pathogens, especially Leptosphaeria maculans (the causal agent of blackleg). Brassica juncea (L.) Czern is an important germplasm resource for canola improvement, due to its good agronomic traits, such as heat and drought tolerance and high blackleg resistance. The present study is the first using genome-wide association studies to identify candidate genes for blackleg resistance in B. juncea based on genome-wide SNPs obtained from the Illumina Infinium 90 K Brassica SNP array. The verification of Rlm6 in B. juncea was performed through a cotyledon infection test. Genotyping 42,914 single nucleotide polymorphisms (SNPs) in a panel of 167 B. juncea lines revealed a total of seven SNPs significantly associated with Rlm6 on chromosomes A07 and B04 in B. juncea. Furthermore, 16 candidate Rlm6 genes were found in these regions, defined as nucleotide binding site leucine-rich-repeat (NLR), leucine-rich repeat RLK (LRR-RLK) and LRR-RLP genes. This study will give insights into the blackleg resistance in B. juncea and facilitate identification of functional blackleg resistance genes which can be used in Brassica breeding.

Linkage mapping and QTL analysis of flowering time using ddRAD sequencing with genotype error correction in Brassica napus.

BACKGROUND: Brassica napus is an important oilseed crop cultivated worldwide. During domestication and breeding of B. napus, flowering time has been a target of selection because of its substantial impact on yield. Here we use double digest restriction-site associated DNA sequencing (ddRAD) to investigate the genetic basis of flowering in B. napus. An F2 mapping population was derived from a cross between an early-flowering spring type and a late-flowering winter type. RESULTS: Flowering time in the mapping population differed by up to 25 days between individuals. High genotype error rates persisted after initial quality controls, as suggested by a genotype discordance of ~ 12% between biological sequencing replicates. After genotype error correction, a linkage map spanning 3981.31 cM and compromising 14,630 single nucleotide polymorphisms (SNPs) was constructed. A quantitative trait locus (QTL) on chromosome C2 was detected, covering eight flowering time genes including FLC. CONCLUSIONS: These findings demonstrate the effectiveness of the ddRAD approach to sample the B. napus genome. Our results also suggest that ddRAD genotype error rates can be higher than expected in F2 populations. Quality filtering and genotype correction and imputation can substantially reduce these error rates and allow effective linkage mapping and QTL analysis.

Genomic comparison of two independent seagrass lineages reveals habitat-driven convergent evolution.

Seagrasses are marine angiosperms that live fully submerged in the sea. They evolved from land plant ancestors, with multiple species representing at least three independent return-to-the-sea events. This raises the question of whether these marine angiosperms followed the same adaptation pathway to allow them to live and reproduce under the hostile marine conditions. To compare the basis of marine adaptation between seagrass lineages, we generated genomic data for Halophila ovalis and compared this with recently published genomes for two members of Zosteraceae, as well as genomes of five non-marine plant species (Arabidopsis, Oryza sativa, Phoenix dactylifera, Musa acuminata, and Spirodela polyrhiza). Halophila and Zosteraceae represent two independent seagrass lineages separated by around 30 million years. Genes that were lost or conserved in both lineages were identified. All three species lost genes associated with ethylene and terpenoid biosynthesis, and retained genes related to salinity adaptation, such as those for osmoregulation. In contrast, the loss of the NADH dehydrogenase-like complex is unique to H. ovalis. Through comparison of two independent return-to-the-sea events, this study further describes marine adaptation characteristics common to seagrass families, identifies species-specific gene loss, and provides molecular evidence for convergent evolution in seagrass lineages.

A chromosome-level genome assembly of Plantago ovata.

Plantago ovata is cultivated for production of its seed husk (psyllium). When wet, the husk transforms into a mucilage with properties suitable for pharmaceutical industries, utilised in supplements for controlling blood cholesterol levels, and food industries for making gluten-free products. There has been limited success in improving husk quantity and quality through breeding approaches, partly due to the lack of a reference genome. Here we constructed the first chromosome-scale reference assembly of P. ovata using a combination of 5.98 million PacBio and 636.5 million Hi-C reads. We also used corrected PacBio reads to estimate genome size and transcripts to generate gene models. The final assembly covers ~ 500 Mb with 99.3% gene set completeness. A total of 97% of the sequences are anchored to four chromosomes with an N50 of ~ 128.87 Mb. The P. ovata genome contains 61.90% repeats, where 40.04% are long terminal repeats. We identified 41,820 protein-coding genes, 411 non-coding RNAs, 108 ribosomal RNAs, and 1295 transfer RNAs. This genome will provide a resource for plant breeding programs to, for example, reduce agronomic constraints such as seed shattering, increase psyllium yield and quality, and overcome crop disease susceptibility.

Identification of candidate genes for LepR1 resistance against Leptosphaeria maculans in Brassica napus.

Utilising resistance (R) genes, such as LepR1, against Leptosphaeria maculans, the causal agent of blackleg in canola (Brassica napus), could help manage the disease in the field and increase crop yield. Here we present a genome wide association study (GWAS) in B. napus to identify LepR1 candidate genes. Disease phenotyping of 104 B. napus genotypes revealed 30 resistant and 74 susceptible lines. Whole genome re-sequencing of these cultivars yielded over 3 million high quality single nucleotide polymorphisms (SNPs). GWAS in mixed linear model (MLM) revealed a total of 2,166 significant SNPs associated with LepR1 resistance. Of these SNPs, 2108 (97%) were found on chromosome A02 of B. napus cv. Darmor bzh v9 with a delineated LepR1_mlm1 QTL at 15.11-26.08 Mb. In LepR1_mlm1, there are 30 resistance gene analogs (RGAs) (13 nucleotide-binding site-leucine rich repeats (NLRs), 12 receptor-like kinases (RLKs), and 5 transmembrane-coiled-coil (TM-CCs)). Sequence analysis of alleles in resistant and susceptible lines was undertaken to identify candidate genes. This research provides insights into blackleg resistance in B. napus and assists identification of the functional LepR1 blackleg resistance gene.

Karyology and Genome Size Analyses of Iranian Endemic Pimpinella (Apiaceae) Species.

Pimpinella species are annual, biennial, and perennial semibushy aromatic plants cultivated for folk medicine, pharmaceuticals, food, and spices. The karyology and genome size of 17 populations of 16 different Pimpinella species collected from different locations in Iran were analyzed for inter-specific karyotypic and genome size variations. For karyological studies, root tips were squashed and painted with a DAPI solution (1 mg/ml). For flow cytometric measurements, fresh leaves of the standard reference (Solanum lycopersicum cv. Stupick, 2C DNA = 1.96 pg) and the Pimpinella samples were stained with propidium iodide. We identified two ploidy levels: diploid (2x) and tetraploid (4x), as well as five metaphase chromosomal counts of 18, 20, 22, 24, and 40. 2n = 24 is reported for the first time in the Pimpinella genus, and the presence of a B-chromosome is reported for one species. The nuclear DNA content ranged from 2C = 2.48 to 2C = 5.50 pg, along with a wide range of genome sizes between 1212.72 and 2689.50 Mbp. The average monoploid genome size and the average value of 2C DNA/chromosome were not proportional to ploidy. There were considerable positive correlations between 2C DNA and total chromatin length and total chromosomal volume. The present study results enable us to classify the genus Pimpinella with a high degree of morphological variation in Iran. In addition, cytological studies demonstrate karyotypic differences between P. anthriscoides and other species of Pimpinella, which may be utilized as a novel identification key to affiliate into a distinct, new genus - Pseudopimpinella.

Genomics reveals the history of a complex plant invasion and improves the management of a biological invasion from the South African-Australian biotic exchange.

Many plants exchanged in the global redistribution of species in the last 200 years, particularly between South Africa and Australia, have become threatening invasive species in their introduced range. Refining our understanding of the genetic diversity and population structure of native and alien populations, introduction pathways, propagule pressure, naturalization, and initial spread, can transform the effectiveness of management and prevention of further introductions. We used 20,221 single nucleotide polymorphisms to reconstruct the invasion of a coastal shrub, Chrysanthemoides monilifera ssp. rotundata (bitou bush) from South Africa, into eastern Australia (EAU), and Western Australia (WAU). We determined genetic diversity and population structure across the native and introduced ranges and compared hypothesized invasion scenarios using Bayesian modeling. We detected considerable genetic structure in the native range, as well as differentiation between populations in the native and introduced range. Phylogenetic analysis showed the introduced samples to be most closely related to the southern-most native populations, although Bayesian analysis inferred introduction from a ghost population. We detected strong genetic bottlenecks during the founding of both the EAU and WAU populations. It is likely that the WAU population was introduced from EAU, possibly involving an unsampled ghost population. The number of private alleles and polymorphic SNPs successively decreased from South Africa to EAU to WAU, although heterozygosity remained high. That bitou bush remains an invasion threat in EAU, despite reduced genetic diversity, provides a cautionary biosecurity message regarding the risk of introduction of potentially invasive species via shipping routes.

Copy Number Variation among Resistance Genes Analogues in Brassica napus.

Copy number variations (CNVs) are defined as deletions, duplications and insertions among individuals of a species. There is growing evidence that CNV is a major factor underlining various autoimmune disorders and diseases in humans; however, in plants, especially oilseed crops, the role of CNVs in disease resistance is not well studied. Here, we investigate the genome-wide diversity and genetic properties of CNVs in resistance gene analogues (RGAs) across eight Brassica napus lines. A total of 1137 CNV events (704 deletions and 433 duplications) were detected across 563 RGAs. The results show CNVs are more likely to occur across clustered RGAs compared to singletons. In addition, 112 RGAs were linked to a blackleg resistance QTL, of which 25 were affected by CNV. Overall, we show that the presence and abundance of CNVs differ between lines, suggesting that in B. napus, the distribution of CNVs depends on genetic background. Our findings advance the understanding of CNV as an important type of genomic structural variation in B. napus and provide a resource to support breeding of advanced canola lines.

Tissue-specific transcriptome profiles identify functional differences key to understanding whole plant response to life in variable salinity.

Plants endure environmental stressors via adaptation and phenotypic plasticity. Studying these mechanisms in seagrasses is extremely relevant as they are important primary producers and functionally significant carbon sinks. These mechanisms are not well understood at the tissue level in seagrasses. Using RNA-seq, we generated transcriptome sequences from tissue of leaf, basal leaf meristem and root organs of Posidonia australis, establishing baseline in situ transcriptomic profiles for tissues across a salinity gradient. Samples were collected from four P. australis meadows growing in Shark Bay, Western Australia. Analysis of gene expression showed significant differences between tissue types, with more variation among leaves than meristem or roots. Gene ontology enrichment analysis showed the differences were largely due to the role of photosynthesis, plant growth and nutrient absorption in leaf, meristem and root organs, respectively. Differential gene expression of leaf and meristem showed upregulation of salinity regulation processes in higher salinity meadows. Our study highlights the importance of considering leaf meristem tissue when evaluating whole-plant responses to environmental change. This article has an associated First Person interview with the first author of the paper.

Genome Analysis of the Broad Host Range Necrotroph Nalanthamala psidii Highlights Genes Associated With Virulence.

Guava wilt disease is caused by the fungus Nalanthamala psidii. The wilt disease results in large-scale destruction of orchards in South Africa, Taiwan, and several Southeast Asian countries. De novo assembly, annotation, and in-depth analysis of the N. psidii genome were carried out to facilitate the identification of characteristics associated with pathogenicity and pathogen evolution. The predicted secretome revealed a range of CAZymes, proteases, lipases and peroxidases associated with plant cell wall degradation, nutrient acquisition, and disease development. Further analysis of the N. psidii carbohydrate-active enzyme profile exposed the broad-spectrum necrotrophic lifestyle of the pathogen, which was corroborated by the identification of putative effectors and secondary metabolites with the potential to induce tissue necrosis and cell surface-dependent immune responses. Putative regulatory proteins including transcription factors and kinases were identified in addition to transporters potentially involved in the secretion of secondary metabolites. Transporters identified included important ABC and MFS transporters involved in the efflux of fungicides. Analysis of the repetitive landscape and the detection of mechanisms linked to reproduction such as het and mating genes rendered insights into the biological complexity and evolutionary potential of N. psidii as guava pathogen. Hence, the assembly and annotation of the N. psidii genome provided a valuable platform to explore the pathogenic potential and necrotrophic lifestyle of the guava wilt pathogen.

Two independent approaches converge to the cloning of a new Leptosphaeria maculans avirulence effector gene, AvrLmS-Lep2.

Brassica napus (oilseed rape, canola) seedling resistance to Leptosphaeria maculans, the causal agent of blackleg (stem canker) disease, follows a gene-for-gene relationship. The avirulence genes AvrLmS and AvrLep2 were described to be perceived by the resistance genes RlmS and LepR2, respectively, present in B. napus 'Surpass 400'. Here we report cloning of AvrLmS and AvrLep2 using two independent methods. AvrLmS was cloned using combined in vitro crossing between avirulent and virulent isolates with sequencing of DNA bulks from avirulent or virulent progeny (bulked segregant sequencing). AvrLep2 was cloned using a biparental cross of avirulent and virulent L. maculans isolates and a classical map-based cloning approach. Taking these two approaches independently, we found that AvrLmS and AvrLep2 are the same gene. Complementation of virulent isolates with this gene confirmed its role in inducing resistance on Surpass 400, Topas-LepR2, and an RlmS-line. The gene, renamed AvrLmS-Lep2, encodes a small cysteine-rich protein of unknown function with an N-terminal secretory signal peptide, which is a common feature of the majority of effectors from extracellular fungal plant pathogens. The AvrLmS-Lep2/LepR2 interaction phenotype was found to vary from a typical hypersensitive response through intermediate resistance sometimes towards susceptibility, depending on the inoculation conditions. AvrLmS-Lep2 was nevertheless sufficient to significantly slow the systemic growth of the pathogen and reduce the stem lesion size on plant genotypes with LepR2, indicating the potential efficiency of this resistance to control the disease in the field.

Genomics Armed With Diversity Leads the Way in Brassica Improvement in a Changing Global Environment.

Meeting the needs of a growing world population in the face of imminent climate change is a challenge; breeding of vegetable and oilseed Brassica crops is part of the race in meeting these demands. Available genetic diversity constituting the foundation of breeding is essential in plant improvement. Elite varieties, land races, and crop wild species are important resources of useful variation and are available from existing genepools or genebanks. Conservation of diversity in genepools, genebanks, and even the wild is crucial in preventing the loss of variation for future breeding efforts. In addition, the identification of suitable parental lines and alleles is critical in ensuring the development of resilient Brassica crops. During the past two decades, an increasing number of high-quality nuclear and organellar Brassica genomes have been assembled. Whole-genome re-sequencing and the development of pan-genomes are overcoming the limitations of the single reference genome and provide the basis for further exploration. Genomic and complementary omic tools such as microarrays, transcriptomics, epigenetics, and reverse genetics facilitate the study of crop evolution, breeding histories, and the discovery of loci associated with highly sought-after agronomic traits. Furthermore, in genomic selection, predicted breeding values based on phenotype and genome-wide marker scores allow the preselection of promising genotypes, enhancing genetic gains and substantially quickening the breeding cycle. It is clear that genomics, armed with diversity, is set to lead the way in Brassica improvement; however, a multidisciplinary plant breeding approach that includes phenotype = genotype × environment × management interaction will ultimately ensure the selection of resilient Brassica varieties ready for climate change.

Modeling first order additive × additive epistasis improves accuracy of genomic prediction for sclerotinia stem rot resistance in canola.

The fungus Sclerotinia sclerotiorum infects hundreds of plant species including many crops. Resistance to this pathogen in canola (Brassica napus L. subsp. napus) is controlled by numerous quantitative trait loci (QTL). For such polygenic traits, genomic prediction may be useful for breeding as it can capture many QTL at once while also considering nonadditive genetic effects. Here, we test application of common regression models to genomic prediction of S. sclerotiorum resistance in canola in a diverse panel of 218 plants genotyped at 24,634 loci. Disease resistance was scored by infection with an aggressive isolate and monitoring over 3 wk. We found that including first-order additive × additive epistasis in linear mixed models (LMMs) improved accuracy of breeding value estimation between 3 and 40%, depending on method of assessment, and correlation between phenotypes and predicted total genetic values by 14%. Bayesian models performed similarly to or worse than genomic relationship matrix-based models for estimating breeding values or overall phenotypes from genetic values. Bayesian ridge regression, which is most similar to the genomic relationship matrix-based approach in the amount of shrinkage it applies to marker effects, was the most accurate of this family of models. This confirms several studies indicating the highly polygenic nature of sclerotinia stem rot resistance. Overall, our results highlight the use of simple epistasis terms for prediction of breeding values and total genetic values for a complex disease resistance phenotype in canola.

QTL Genetic Mapping Study for Traits Affecting Meal Quality in Winter Oilseed Rape (Brassica Napus L.).

Rapeseed (Brassica napus L.) meal is an important source of protein, but the presence of anti-nutritional compounds, such as fibre and glucosinolates, still limits its use as a livestock feed. Understanding the genetic basis of seed fibre biosynthesis would help to manipulate its content in seeds of oilseed rape. Here, we applied high-resolution skim genotyping by sequencing (SkimGBS) and characterised 187,835 single-nucleotide polymorphism (SNP) markers across a mapping population subsequently used for a genetic mapping study (R/qtl). This approach allowed the identification of 11 stable QTL related to seed quality traits and led to the identification of potential functional genes underlying these traits. Among these, key genes with a known role in carbohydrate metabolic process, cell wall, lignin, and flavonoid biosynthesis, including cellulase GH5, TT10/LAC15, TT4, and SUC2, were found. This study furthers the understanding of the molecular mechanisms underlying seed fibre content and provides new markers for molecular breeding in B. napus.

Genotyping for Species Identification and Diversity Assessment Using Double-Digest Restriction Site-Associated DNA Sequencing (ddRAD-Seq).

Genotyping-by-sequencing (GBS) is a powerful approach for studying the genetic diversity of legume species. By using restriction enzymes or other methods to generate a reduced representation of the genome for sequencing, GBS can provide genome-wide single nucleotide polymorphisms (SNP) for diversity analysis at high throughput and low cost. Here we describe a novel double-digest restriction site-associated DNA sequencing (ddRAD-seq) approach. We also describe the downstream bioinformatic analysis of the sequencing data, including alignment to a reference genome, de novo assembly, SNP calling, phylogenetic analysis, and structure analysis.