Lars G. Kamphuis

SSR analysis of the Medicago truncatula SARDI core collection reveals substantial diversity and unusual genotype dispersal throughout the Mediterranean basin

The world’s oldest and largest Medicago truncatula collection is housed at the South Australian Research and Development Institute (SARDI). We used six simple sequence repeat (SSR) loci to analyse the genetic diversity and relationships between randomly selected individuals from 192 accessions in the core collection. M. truncatula is composed of three subspecies (ssp.): ssp. truncatula, ssp. longeaculeata, and ssp. tricycla. Analysis at the level of six SSR loci supports the concept of ssp. tricycla, all the samples of which showed unique alleles at two loci. Contingency Chi-squared tests were significant between ssp. tricycla and ssp. truncatula at four loci, suggesting a barrier to gene flow between these subspecies. In accessions defined as ssp. longeaculeata, no unique allelic distribution or diagnostic sizes were observed, suggesting this apparent ssp. is a morphological variant of ssp. truncatula. The data also suggest M. truncatula that exhibits unusually wide genotype dispersal throughout its native Mediterranean region, possibly due to animal and trade-related movements. Our results showed the collection to be highly diverse, exhibiting an average of 25 SSR alleles per locus, with over 90% of individuals showing discrete genotypes. The rich diversity of the SARDI collection provides an invaluable resource for studying natural allelic variation of M. truncatula. To efficiently exploit the variation in the SARDI collection, we have defined a subset of accessions (n=61) that maximises the diversity.

Two alternative recessive quantitative trait loci influence resistance to spring black stem and leaf spot in Medicago truncatula

BackgroundKnowledge of the genetic basis of plant resistance to necrotrophic pathogens is incomplete and has been characterised in relatively few pathosystems. In this study, the cytology and genetics of resistance to spring black stem and leaf spot caused by Phoma medicaginis, an economically important necrotrophic pathogen of Medicago spp., was examined in the model legume M. truncatula.ResultsMacroscopically, the resistant response of accession SA27063 was characterised by small, hypersensitive-like spots following inoculation while the susceptible interaction with accessions A17 and SA3054 showed necrotic lesions and spreading chlorosis. No unique cytological differences were observed during early infection (<48 h) between the resistant and susceptible genotypes, except pathogen growth was restricted to one or a few host cells in SA27063. In both interactions reactive oxygen intermediates and phenolic compounds were produced, and cell death occurred. Two F2 populations segregating for resistance to spring black stem and leaf spot were established between SA27063 and the two susceptible accessions, A17 and SA3054. The cross between SA27063 and A17 represented a wider cross than between SA27063 and SA3054, as evidenced by higher genetic polymorphism, reduced fertility and aberrant phenotypes of F2 progeny. In the SA27063 × A17 F2 population a highly significant quantitative trait locus (QTL, LOD = 7.37; P < 0.00001) named resistance to the necrotroph Phomamedicaginis one (rnpm1) genetically mapped to the top arm of linkage group 4 (LG4). rnpm1 explained 33.6% of the phenotypic variance in the populations response to infection depicted on a 1–5 scale and was tightly linked to marker AW256637. A second highly significant QTL (LOD = 6.77; P < 0.00001), rnpm2, was located on the lower arm of LG8 in the SA27063 × SA3054 map. rnpm2 explained 29.6% of the phenotypic variance and was fine mapped to a 0.8 cM interval between markers h2_16a6a and h2_21h11d. rnpm1 is tightly linked to a cluster of Toll/Interleukin1 receptor-nucleotide binding site-leucine-rich repeat (TIR-NBS-LRR) genes and disease resistance protein-like genes, while no resistance gene analogues (RGAs) are apparent in the genomic sequence of the reference accession A17 at the rnpm2 locus.ConclusionThe induction of defence responses and cell death in the susceptible interaction following infection by P. medicaginis suggested this pathogen is not negatively affected by these responses and may promote them. A QTL for resistance was revealed in each of two populations derived from crosses between a resistant accession and two different susceptible accessions. Both loci are recessive in nature, and the simplest explanation for the existence of two separate QTLs is the occurrence of host genotype-specific susceptibility loci that may interact with undetermined P. medicaginis virulence factors.

Identification of Sources of Resistance to Phoma medicaginis Isolates in Medicago truncatula SARDI Core Collection Accessions, and Multigene Differentiation of Isolates

ABSTRACT Phoma medicaginis is a necrotrophic fungal pathogen, commonly found infecting the annual medic Medicago truncatula. To differentiate eight P. medicaginis isolates, five gene regions were examined: actin, beta-tubulin, calmodulin, translation elongation factor 1-alpha (EF-1alpha), and the internal transcribed spacer ribosomal DNA. Sequence comparisons showed that specimens isolated from M. truncatula in Western Australia formed a group that was consistently different from, but allied to, a P. medicaginis var. medicaginis type specimen. EF-1alpha contained a hyper-variable 55-bp repeat unit, which forms the basis of a rapid polymerase chain reaction-based method of reliably distinguishing isolates. Characterization of three isolates showed that all exhibited a narrow host range, causing disease only in M. sativa and M. truncatula among eight commonly cultivated legume species sampled. Infection of 86 M. truncatula single-seeded accessions showed a continuous distribution in disease phenotypes, with the majority of accessions susceptible. On a 1-to-5 disease reaction scale increasing in severity, individual fungal isolates showed means of 2.6 to 3.2, and scores ranged from 1 to 4.8 among accessions. The results presented here suggest that M. truncatula harbors specific and diverse sources of resistance to individual P. medicaginis genotypes.

Transcriptome sequencing of different narrow-leafed lupin tissue types provides a comprehensive uni-gene assembly and extensive gene-based molecular markers

Narrow-leafed lupin (NLL; Lupinus angustifolius L.) is an important grain legume crop that is valuable for sustainable farming and is becoming recognized as a human health food. NLL breeding is directed at improving grain production, disease resistance, drought tolerance and health benefits. However, genetic and genomic studies have been hindered by a lack of extensive genomic resources for the species. Here, the generation, de novo assembly and annotation of transcriptome datasets derived from five different NLL tissue types of the reference accession cv. Tanjil are described. The Tanjil transcriptome was compared to transcriptomes of an early domesticated cv. Unicrop, a wild accession P27255, as well as accession 83A:476, together being the founding parents of two recombinant inbred line (RIL) populations. In silico predictions for transcriptome-derived gene-based length and SNP polymorphic markers were conducted and corroborated using a survey assembly sequence for NLL cv. Tanjil. This yielded extensive indel and SNP polymorphic markers for the two RIL populations. A total of 335 transcriptome-derived markers and 66 BAC-end sequence-derived markers were evaluated, and 275 polymorphic markers were selected to genotype the reference NLL 83A:476 × P27255 RIL population. This significantly improved the completeness, marker density and quality of the reference NLL genetic map.

Development of genomic resources for the narrow-leafed lupin (Lupinus angustifolius): construction of a bacterial artificial chromosome (BAC) library and BAC-end sequencing

BackgroundLupinus angustifolius L, also known as narrow-leafed lupin (NLL), is becoming an important grain legume crop that is valuable for sustainable farming and is becoming recognised as a potential human health food. Recent interest is being directed at NLL to improve grain production, disease and pest management and health benefits of the grain. However, studies have been hindered by a lack of extensive genomic resources for the species.ResultsA NLL BAC library was constructed consisting of 111,360 clones with an average insert size of 99.7 Kbp from cv Tanjil. The library has approximately 12 × genome coverage. Both ends of 9600 randomly selected BAC clones were sequenced to generate 13985 BAC end-sequences (BESs), covering approximately 1% of the NLL genome. These BESs permitted a preliminary characterisation of the NLL genome such as organisation and composition, with the BESs having approximately 39% G:C content, 16.6% repetitive DNA and 5.4% putative gene-encoding regions. From the BESs 9966 simple sequence repeat (SSR) motifs were identified and some of these are shown to be potential markers.ConclusionsThe NLL BAC library and BAC-end sequences are powerful resources for genetic and genomic research on lupin. These resources will provide a robust platform for future high-resolution mapping, map-based cloning, comparative genomics and assembly of whole-genome sequencing data for the species.

A comprehensive draft genome sequence for lupin (Lupinus angustifolius), an emerging health food: insights into plant-microbe interactions and legume evolution.

Summary Lupins are important grain legume crops that form a critical part of sustainable farming systems, reducing fertilizer use and providing disease breaks. It has a basal phylogenetic position relative to other crop and model legumes and a high speciation rate. Narrow‐leafed lupin (NLL; Lupinus angustifolius L.) is gaining popularity as a health food, which is high in protein and dietary fibre but low in starch and gluten‐free. We report the draft genome assembly (609 Mb) of NLL cultivar Tanjil, which has captured >98% of the gene content, sequences of additional lines and a dense genetic map. Lupins are unique among legumes and differ from most other land plants in that they do not form mycorrhizal associations. Remarkably, we find that NLL has lost all mycorrhiza‐specific genes, but has retained genes commonly required for mycorrhization and nodulation. In addition, the genome also provided candidate genes for key disease resistance and domestication traits. We also find evidence of a whole‐genome triplication at around 25 million years ago in the genistoid lineage leading to Lupinus. Our results will support detailed studies of legume evolution and accelerate lupin breeding programmes.

Identification of potential early regulators of aphid resistance in Medicago truncatula via transcription factor expression profiling.

*Resistance to aphids has been identified in a number of plant species, yet the molecular mechanisms underlying aphid resistance remain largely unknown. *Using high-throughput quantitative real-time PCR technology, the transcription profiles of 752 putative Medicago truncatula transcription factor genes were analysed in a pair of susceptible and resistant closely related lines of M. truncatula following 6 and 12 h of bluegreen aphid (Acyrthosiphon kondoi) infestation. *Eighty-two transcription factor genes belonging to 30 transcription factor families were responsive to bluegreen aphid infestation. More transcription factor genes were responsive in the resistant interaction than in the susceptible interaction; of the 36 genes that were induced at 6 and/or 12 h, 32 were induced only in the resistant interaction. Bluegreen aphid-induced expression of a subset of these genes was correlated with the presence of AKR, a single dominant gene conferring resistance to bluegreen aphids. Similar transcription factor expression patterns of this subset were associated with bluegreen aphid resistance in other M. truncatula genetic backgrounds, as well as with resistance to pea aphid (Acyrthosiphon pisum). *Our results suggest that these transcription factors are among the early aphid-responsive genes in resistant plants, and may play important roles in resistance to multiple aphid species.

The essential role of genetic resources in narrow-leafed lupin improvement

Abstract. The narrow-leafed lupin (Lupinus angustifolius L.) is a legume with much to offer to agriculture and human wellbeing through its adaptation to nitrogen- and phosphorus-deficient, acid, sandy soils, and production of nutritious, very low glycemic index grain with manifold health benefits. However, the industry has exploited only a small fraction of the genetic and adaptive diversity of the species, reflecting a short and fragmented domestication history. Given declining global production, unlocking the potential residing in untapped sources of genetic diversity to maximise yield and value is critical for the future of the crop. To this end, a wide range of genetic resources is under evaluation. The Australian Lupin Collection comprises almost 4600 diverse, mostly wild accessions, many of which have been genotyped using DArT (Diversity Array Technology) markers, and collection sites characterised to facilitate ecophysiology of contrasting material. Additional exotic genetic resources include recombinant inbred line and mutant populations, as well as inter-specific crosses. These resources are being used to investigate specific adaptation and genetic and molecular control of key traits, all of which will be expedited by current efforts to provide a reference genome sequence for L. angustifolius. Genetic base broadening is the current breeding focus, combining distantly related wild and domestic material with elite cultivars in double-backcrosses or topcrosses, with dramatic effects on yield. In future this will be complemented by marker-based, targeted trait introgression to improve narrow-leafed lupin adaptation, quality/value, and fit into the farming system.

Comparative genomics and prediction of conditionally dispensable sequences in legume–infecting Fusarium oxysporum formae speciales facilitates identification of candidate effectors

BackgroundSoil-borne fungi of the Fusarium oxysporum species complex cause devastating wilt disease on many crops including legumes that supply human dietary protein needs across many parts of the globe. We present and compare draft genome assemblies for three legume-infecting formae speciales (ff. spp.): F. oxysporum f. sp. ciceris (Foc-38-1) and f. sp. pisi (Fop-37622), significant pathogens of chickpea and pea respectively, the world’s second and third most important grain legumes, and lastly f. sp. medicaginis (Fom-5190a) for which we developed a model legume pathosystem utilising Medicago truncatula.ResultsFocusing on the identification of pathogenicity gene content, we leveraged the reference genomes of Fusarium pathogens F. oxysporum f. sp. lycopersici (tomato-infecting) and F. solani (pea-infecting) and their well-characterised core and dispensable chromosomes to predict genomic organisation in the newly sequenced legume-infecting isolates. Dispensable chromosomes are not essential for growth and in Fusarium species are known to be enriched in host-specificity and pathogenicity-associated genes. Comparative genomics of the publicly available Fusarium species revealed differential patterns of sequence conservation across F. oxysporum formae speciales, with legume-pathogenic formae speciales not exhibiting greater sequence conservation between them relative to non-legume-infecting formae speciales, possibly indicating the lack of a common ancestral source for legume pathogenicity. Combining predicted dispensable gene content with in planta expression in the model legume-infecting isolate, we identified small conserved regions and candidate effectors, four of which shared greatest similarity to proteins from another legume-infecting ff. spp.ConclusionsWe demonstrate that distinction of core and potential dispensable genomic regions of novel F. oxysporum genomes is an effective tool to facilitate effector discovery and the identification of gene content possibly linked to host specificity. While the legume-infecting isolates didn’t share large genomic regions of pathogenicity-related content, smaller regions and candidate effector proteins were highly conserved, suggesting that they may play specific roles in inducing disease on legume hosts.

Plant-aphid interactions with a focus on legumes

Sap-sucking insects such as aphids cause substantial yield losses in agriculture by draining plant nutrients as well as vectoring viruses. The main method of control in agriculture is through the application of insecticides. However, aphids rapidly evolve mechanisms to detoxify these, so there is a need to develop durable plant resistance to these damaging insect pests. The focus of this review is on aphid interactions with legumes, but work on aphid interactions with other plants, particularly Arabidopsis and tomato is also discussed. This review covers advances on the plant side of the interaction, including the identification of major resistance genes and quantitative trait loci conferring aphid resistance in legumes, basal and resistance gene mediated defence signalling following aphid infestation and the role of specialised metabolites. On the aphid side of the interaction, this review covers what is known about aphid effector proteins and aphid detoxification enzymes. Recent advances in these areas have provided insight into mechanisms underlying resistance to aphids and the strategies used by aphids for successful infestations and have significant impacts for the delivery of durable resistance to aphids in legume crops.