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In a cross-continental research initiative, including researchers working in Australia and Denmark, and based on joint external funding by a 3-year grant from the Novo Nordisk Foundation, we have used DNA sequencing, extensive chemical profiling and molecular networking analyses across the entire Eremophila genus to provide new knowledge on the presence of natural products and their bioactivities using polypharmocological screens. Sesquiterpenoids, diterpenoids and dimers of branched-chain fatty acids with previously unknown chemical structures were identified. The collection of plant material from the Eremophila genus was carried out according to a 'bioprospecting agreement' with the Government of Western Australia. We recognize that several Eremophila species hold immense cultural significance to Australia's First Peoples. In spite of our best intentions to ensure that new knowledge gained about the genus Eremophila and any potential future benefits are shared in an equitable manner, in accordance with the Nagoya Protocol, we encounter serious dilemmas and potential conflicts in making benefit sharing with Australia's First Peoples a reality., (© 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Eremophila is the largest genus in the plant tribe Myoporeae (Scrophulariaceae) and exhibits incredible morphological diversity across the Australian continent. The Australian Aboriginal Peoples recognize many Eremophila species as important sources of traditional medicine, the most frequently used plant parts being the leaves. Recent phylogenetic studies have revealed complex evolutionary relationships between Eremophila and related genera in the tribe. Unique and structurally diverse metabolites, particularly diterpenoids, are also a feature of plants in this group. To assess the full dimension of the chemical space of the tribe Myoporeae, we investigated the metabolite diversity in a chemo-evolutionary framework applying a combination of molecular phylogenetic and state-of-the-art computational metabolomics tools to build a dataset involving leaf samples from a total of 291 specimens of Eremophila and allied genera. The chemo-evolutionary relationships are expounded into a systematic context by integration of information about leaf morphology (resin and hairiness), environmental factors (pollination and geographical distribution), and medicinal properties (traditional medicinal uses and antibacterial studies), augmenting our understanding of complex interactions in biological systems., (© 2021 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Bean yellow mosaic virus (BYMV), genus Potyvirus, has an extensive natural host range encompassing both dicots and monocots. Its phylogenetic groups were considered to consist of an ancestral generalist group and six specialist groups derived from this generalist group during plant domestication. Recombination was suggested to be playing a role in BYMV's evolution towards host specialization. However, in subsequent phylogenetic analysis of whole genomes, group names based on the original hosts of isolates within each of them were no longer supported. Also, nine groups were found and designated I-IX. Recombination analysis was conducted on the complete coding regions of 33 BYMV genomes and two genomes of the related Clover yellow vein virus (CYVV). This analysis found evidence for 12 firm recombination events within BYMV phylogenetic groups I-VI, but none within groups VII-IX or CYVV. The greatest numbers of recombination events within a sequence (two or three each) occurred in four groups, three which formerly constituted the single ancestral generalist group (I, II and IV), and group VI. The individual sequences in groups III and V had one event each. These findings with whole genomes are consistent with recombination being associated with expanding host ranges, and call into question the proposed role of recombination in the evolution of BYMV, where it was previously suggested to play a role in host specialization. Instead, they (i) indicate that recombination explains the very broad natural host ranges of the three BYMV groups which infect both monocots and dicots (I, II, IV), and (ii) suggest that the three groups with narrow natural host ranges (III, V, VI) which also showed recombination now have the potential to reduce host specificity and broaden their natural host ranges.

Next generation sequencing is quickly emerging as the go-to tool for plant virologists when sequencing whole virus genomes, and undertaking plant metagenomic studies for new virus discoveries. This study aims to compare the genomic and biological properties of Bean yellow mosaic virus (BYMV) (genus Potyvirus), isolates from Lupinus angustifolius plants with black pod syndrome (BPS), systemic necrosis or non-necrotic symptoms, and from two other plant species. When one Clover yellow vein virus (ClYVV) (genus Potyvirus) and 22 BYMV isolates were sequenced on the Illumina HiSeq2000, one new ClYVV and 23 new BYMV sequences were obtained. When the 23 new BYMV genomes were compared with 17 other BYMV genomes available on Genbank, phylogenetic analysis provided strong support for existence of nine phylogenetic groupings. Biological studies involving seven isolates of BYMV and one of ClYVV gave no symptoms or reactions that could be used to distinguish BYMV isolates from L. angustifolius plants with black pod syndrome from other isolates. Here, we propose that the current system of nomenclature based on biological properties be replaced by numbered groups (I-IX). This is because use of whole genomes revealed that the previous phylogenetic grouping system based on partial sequences of virus genomes and original isolation hosts was unsustainable. This study also demonstrated that, where next generation sequencing is used to obtain complete plant virus genomes, consideration needs to be given to issues regarding sample preparation, adequate levels of coverage across a genome and methods of assembly. It also provided important lessons that will be helpful to other plant virologists using next generation sequencing in the future.

Black pod syndrome (BPS) causes devastating losses in Lupinus angustifolius (narrow-leafed lupin) crops in Australia, and infection with Bean yellow mosaic virus (BYMV) was suggested as a possible cause. In 2011, an end-of-growing-season survey in which L. angustifolius plants with BPS were collected from six locations in southwestern Australia was done. Tissue samples from different positions on each of these symptomatic plants were tested for BYMV and generic potyvirus by enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction (RT-PCR). Detection was most reliable when RT-PCR with generic potyvirus primers was used on tissue taken from the main stem of the plant just below the black pods. Partial coat protein nucleotide sequences from eight isolates from BPS-symptomatic L. angustifolius plants all belonged to the BYMV general phylogenetic group. An initial glasshouse experiment revealed that mechanical inoculation of L. angustifolius plants with BYMV after pods had formed caused pods to turn black. This did not occur when the plants were inoculated before this growth stage (at first flowering) because BYMV infection caused plant death. A subsequent experiment in which plants were inoculated at eight different growth stages confirmed that BPS was only induced when L. angustifolius plants were inoculated after first flowering, when pods had formed. Thus, BYMV was isolated from symptomatic L. angustifolius survey samples, inoculated to and maintained in culture hosts, inoculated to healthy L. angustifolius test plants inducing BPS, and then successfully reisolated from them. As such, Koch's postulates were fulfilled for the hypothesis that late infection with BYMV causes BPS in L. angustifolius plants.

Hardenbergia mosaic virus (HarMV), genus Potyvirus, belongs to the bean common mosaic virus (BCMV) potyvirus lineage found only in Australia. The original host of HarMV, Hardenbergia comptoniana, family Fabaceae, is indigenous to the South-West Australian Floristic Region (SWAFR), where Lupinus spp. are grown as introduced grain legume crops, and exist as naturalised weeds. Two plants of H. comptoniana and one of Lupinus cosentinii, each with mosaic and leaf deformation symptoms, were sampled from a small patch of disturbed vegetation at an ancient ecosystem-recent agroecosystem interface. Potyvirus infection was detected in all three samples by ELISA and RT-PCR. After sequencing on an Illumina HiSeq 2000, three complete and two nearly complete HarMV genomes from H. comptoniana and one complete HarMV genome from L. cosentinii were obtained. Phylogenetic analysis which compared (i) the four new complete genomes with the three HarMV genomes on Genbank (two of which were identical), and (ii) coat protein (CP) genes from the six new genomes with the 38 HarMV CP sequences already on Genbank, revealed that three of the complete and one of the nearly complete new genomes were in HarMV clade I, one of the complete genomes in clade V and one nearly complete genome in clade VI. The complete HarMV genome from L. cosentinii differed by only eight nucleotides from one of the HarMV clade I genomes from a nearby H. comptoniana plant, with only one of these nucleotide changes being non-synonymous. Pairwise comparison between all the complete HarMV genomes revealed nucleotide identities ranging between 82.2% and 100%. Recombination analysis revealed evidence of two recombination events amongst the six complete genomes. This study provides the first report of HarMV naturally infecting L. cosentinii and the first example for the SWAFR of virus emergence from a native plant species to invade an introduced plant species., (Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.)

Selection for phomopsis stem blight disease (PSB) resistance is one of the key objectives in lupin (Lupinus angustifolius L.) breeding programs. A cross was made between cultivar Tanjil (resistant to PSB) and Unicrop (susceptible). The progeny was advanced into F(8) recombinant inbred lines (RILs). The RIL population was phenotyped for PSB disease resistance. Twenty plants from the RIL population representing disease resistance and susceptibility was subjected to next-generation sequencing (NGS)-based restriction site-associated DNA sequencing on the NGS platform Solexa HiSeq2000, which generated 7,241 single nucleotide polymorphisms (SNPs). Thirty-three SNP markers showed the correlation between the marker genotypes and the PSB disease phenotype on the 20 representative plants, which were considered as candidate markers linked to a putative R gene for PSB resistance. Seven candidate markers were converted into sequence-specific PCR markers, which were designated as PhtjM1, PhtjM2, PhtjM3, PhtjM4, PhtjM5, PhtjM6 and PhtjM7. Linkage analysis of the disease phenotyping data and marker genotyping data on a F(8) population containing 187 RILs confirmed that all the seven converted markers were associated with the putative R gene within the genetic distance of 2.1 CentiMorgan (cM). One of the PCR markers, PhtjM3, co-segregated with the R gene. The seven established PCR markers were tested in the 26 historical and current commercial cultivars released in Australia. The numbers of "false positives" (showing the resistance marker allele band but lack of the putative R gene) for each of the seven PCR markers ranged from nil to eight. Markers PhtjM4 and PhtjM7 are recommended in marker-assisted selection for PSB resistance in the Australian national lupin breeding program due to its wide applicability on breeding germplasm and close linkage to the putative R gene. The results demonstrated that application of NGS technology is a rapid and cost-effective approach in development of markers for molecular plant breeding.

In contrast to most widespread broad-acre crops, the narrow-leafed lupin (Lupinus angustifolius L.) was domesticated very recently, in breeding programmes isolated in both space and time. Whereas domestication was initiated in Central Europe in the early twentieth century, the crop was subsequently industrialized in Australia, which now dominates world production. To investigate the ramifications of these bottlenecks, the genetic diversity of wild (n = 1,248) and domesticated populations (n = 95) was characterized using diversity arrays technology, and adaptation studied using G × E trials (n = 31) comprising all Australian cultivars released from 1967 to 2004 (n = 23). Principal coordinates analysis demonstrates extremely limited genetic diversity in European and Australian breeding material compared to wild stocks. AMMI analysis indicates that G × E interaction is a minor, albeit significant effect, dominated by strong responses to local, Western Australian (WA) optima. Over time Australian cultivars have become increasingly responsive to warm, intermediate rainfall environments in the northern WA grainbelt, but much less so to cool vegetative phase eastern environments, which have considerably more yield potential. G × E interaction is well explained by phenology, and its interaction with seasonal climate, as a result of varying vernalization responses. Yield differences are minimized when vegetative phase temperatures fully satisfy the vernalization requirement (typical of eastern Australia), and maximized when they do not (typical of WA). In breeding for WA optima, the vernalization response has been eliminated and there has been strong selection for terminal drought avoidance through early phenology, which limits yield potential in longer season eastern environments. Conversely, vernalization-responsive cultivars are more yield-responsive in the east, where low temperatures moderately extend the vegetative phase. The confounding of phenology and vernalization response limits adaptation in narrow-leafed lupin, isolates breeding programmes, and should be eliminated by widening the flowering time range in a vernalization-unresponsive background. Concomitantly, breeding strategies that will widen the genetic base of the breeding pool in an ongoing manner should be initiated.

Selection for anthracnose disease resistance is one of the major objectives in lupin breeding programs. The aim of this study was to develop a molecular marker linked to a gene conferring anthracnose resistance in narrow-leafed lupin (Lupinus angustifolius L.), which can be widely used for MAS in lupin breeding. A F(8)derived RIL population from a cross between cultivar Tanjil (resistant to anthracnose) and Unicrop (susceptible) was used for marker development. DNA fingerprinting was conducted on 12 representative plants by combining the AFLP method with primers designed based on conserved sequences of plant disease resistance genes. A co-dominant candidate marker was detected from a DNA fingerprint. The candidate marker was cloned, sequenced, and converted into a sequence-specific, simple PCR based marker. Linkage analysis based on a segregating population consisting of 184 RILs suggested that the marker, designated as AntjM2, is located 2.3 cM away from the R gene conferring anthracnose resistance in L. angustifolius. The marker has now being implemented for MAS in the Australian national lupin breeding program.

A mapping population of F(8)derived recombinant inbred lines (RILs) was established from a cross between a domesticated breeding line 83A:476 and a wild type P27255 in narrow-leaf lupin (Lupinus angustifolius L.). The parents together with the 89 RILs were subjected to DNA fingerprinting using microsatellite-anchored fragment length polymorphism (MFLP) to rapidly generate DNA markers to construct a linkage map. Five hundred and twenty two unique markers of which 21% were co-dominant, were generated and mapped. Phenotypic data for the domestication traits: mollis (soft seeds), leucospermus (white flower and seed colour); Lentus (reduced pod-shattering), iucundis (low alkaloid), Ku (early flowering) and moustache pattern on seed coats; were included. Three to 7 molecular markers were identified within 5 cM of each of these domestication genes. The anthracnose resistance gene Lanr1 was also mapped. Linkage groups were constructed using MapManager version QTXb20, resulting in 21 linkage groups consisting of 7 or more markers. The total map length was 1543 cM, with an average distance of 3.4 cM between adjacent markers. This is the first published map for a lupin species. The map can be exploited for marker assisted selection for genetic improvement in lupin breeding programs.