Genome assembly and transcriptomic analysis to elucidate the ability of Nasonovia ribisnigri to break host plant resistance.

Aphid genomic resources enable the study of complex life history traits and provide information on vector biology, host adaption and speciation. The currant-lettuce aphid (Nasonovia ribisnigri (Hemiptera: Aphididae) (Mosley)) is a cosmopolitan pest of outdoor lettuce (Lactuca sativa (Asterales: Asteraceae) (Linnaeus)). Until recently, the use of resistant cultivars was an effective method for managing N. ribisnigri. A resistant cultivar containing a single gene (Nr-locus), introduced in the 1980s, conferred complete resistance to feeding. Overreliance of this Nr-locus in lettuce resulted in N. ribisnigri's ability to break resistance mechanism, with first reports during 2003. Our work attempts to understand which candidate gene(s) are associated with this resistance-breaking mechanism. We present two de novo draft assembles for N. ribisnigri genomes, corresponding to both avirulent (Nr-locus susceptible) and virulent (Nr-locus resistant) biotypes. Changes in gene expression of the two N. ribisnigri biotypes were investigated using transcriptomic analyses of RNA-sequencing (RNA-seq) data to understand the potential mechanisms of resistance to the Nr-locus in lettuce. The draft genome assemblies were 94.2% and 91.4% complete for the avirulent and virulent biotypes, respectively. Out of the 18,872 differentially expressed genes, a single gene/locus was identified in N. ribisnigri that was shared between two resistant-breaking biotypes. This locus was further explored and validated in Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) experiments and has predicted localisations in both the cytoplasm and nucleus. This is the first study to provide evidence that a single gene/locus is likely responsible for the ability of N. ribisnigri to overcome the Nr-locus resistance in the lettuce host.

Tipburn resilience in lettuce (Lactuca spp.) - the importance of germplasm resources and production system-specific assays.

BACKGROUND: Tipburn is a physiological disorder of lettuce (Lactuca spp.). It causes discoloration and collapse of leaf margins, leading to unsaleable crops in both protected (glasshouse, hydroponic) and outdoor production systems. The occurrence of tipburn is hard to predict and is sensitive to environmental conditions. Phenotyping for tipburn resilience requires diverse germplasm resources and, to date, limited material has been investigated for this condition. RESULTS: Using a Lactuca diversity fixed foundation set (DFFS) under glasshouse conditions, we identified a significant (P < 0.001) genotypic effect on tipburn resilience across both the entire population and across lines belonging to the cultivated species L. sativa alone. Latuca sativa lines exhibited significantly (P < 0.05) higher average tipburn severity than those belonging to the wild species L. saligna, L. serriola, and L. virosa but we were able to identify both cultivated and wild tipburn-resilient lines. Leaf morphology factors, which included pigmentation, width, and serration, also significantly (P < 0.05) influenced tipburn resilience. Using a recombinant inbred line (RIL) mapping population derived from two DFFS lines, different small-effect quantitative trait loci (QTLs) accounting for 12.3% and 25.2% of total tipburn variation were identified in glasshouse and field conditions, respectively. CONCLUSIONS: These results reflect the advantages of phenotyping under production-system-specific conditions for the examination of environmentally sensitive traits and highlight genetic markers and germplasm resources for the development of tipburn resilient lines for use in both protected and outdoor lettuce production. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Unravelling effects of red/far-red light on nutritional quality and the role and mechanism in regulating lycopene synthesis in postharvest cherry tomatoes.

The main goal of this study was to explore the role of red/far-red light in the preservation of postharvest quality in cherry tomato fruits and the mechanism of red/far-red light in regulation of lycopene synthesis. Results showed that red/far-red light irradiation inhibited weight loss and promoted colour change during storage, and it also increased the content of lycopene and ß-carotene compared to control. Gene PSY, ZDS and LCY-b were overexpressed in fruits treated with red/far-red light during 33 days' storage compared to control. The analysis of genes involved in red/far-red light absorbance (PHYA and PHYB) and mediation (HY5 and PIF3), and fruit ripening (ACS2 and RIN) suggests that red/far-red light promote lycopene accumulation through phytochrome-mediated signalling pathway to induce HY5. Elevated HY5 could either directly bind to PSY or promote the expression of ACS2 to induce RIN through MADS-loop to enhanced lycopene content.

A review of sources of resistance to turnip yellows virus (TuYV) in Brassica species.

Turnip yellows virus (TuYV; previously known as beet western yellows virus) causes major diseases of Brassica species worldwide resulting in severe yield-losses in arable and vegetable crops. It has also been shown to reduce the quality of vegetables, particularly cabbage where it causes tip burn. Incidences of 100% have been recorded in commercial crops of winter oilseed rape (Brassica napus) and vegetable crops (particularly Brassica oleracea) in Europe. This review summarises the known sources of resistance to TuYV in B. napus (AACC genome), Brassica rapa (AA genome) and B. oleracea (CC genome). It also proposes names for the quantitative trait loci (QTLs) responsible for the resistances, Turnip Yellows virus Resistance (TuYR), that have been mapped to at least the chromosome level in the different Brassica species. There is currently only one known source of resistance deployed commercially (TuYR1). This resistance is said to have originated in B. rapa and was introgressed into the A genome of oilseed rape via hybridisation with B. oleracea to produce allotetraploid (AACC) plants that were then backcrossed into oilseed rape. It has been utilised in the majority of known TuYV-resistant oilseed rape varieties. This has placed significant selection pressure for resistance-breaking mutations arising in TuYV. Further QTLs for resistance to TuYV (TuYR2-TuYR9) have been mapped in the genomes of B. napus, B. rapa and B. oleracea and are described here. QTLs from the latter two species have been introgressed into allotetraploid plants, providing for the first time, combined resistance from both the A and the C genomes for deployment in oilseed rape. Introgression of these new resistances into commercial oilseed rape and vegetable brassicas can be accelerated using the molecular markers that have been developed. The deployment of these resistances should lessen selection pressure for resistance-breaking isolates of TuYV and thereby prolong the effectiveness of each other and extant resistance.

Crop management shapes the diversity and activity of DNA and RNA viruses in the rhizosphere.

BACKGROUND: The rhizosphere is a hotspot for microbial activity and contributes to ecosystem services including plant health and biogeochemical cycling. The activity of microbial viruses, and their influence on plant-microbe interactions in the rhizosphere, remains undetermined. Given the impact of viruses on the ecology and evolution of their host communities, determining how soil viruses influence microbiome dynamics is crucial to build a holistic understanding of rhizosphere functions. RESULTS: Here, we aimed to investigate the influence of crop management on the composition and activity of bulk soil, rhizosphere soil, and root viral communities. We combined viromics, metagenomics, and metatranscriptomics on soil samples collected from a 3-year crop rotation field trial of oilseed rape (Brassica napus L.). By recovering 1059 dsDNA viral populations and 16,541 ssRNA bacteriophage populations, we expanded the number of underexplored Leviviricetes genomes by > 5 times. Through detection of viral activity in metatranscriptomes, we uncovered evidence of "Kill-the-Winner" dynamics, implicating soil bacteriophages in driving bacterial community succession. Moreover, we found the activity of viruses increased with proximity to crop roots, and identified that soil viruses may influence plant-microbe interactions through the reprogramming of bacterial host metabolism. We have provided the first evidence of crop rotation-driven impacts on soil microbial communities extending to viruses. To this aim, we present the novel principal of "viral priming," which describes how the consecutive growth of the same crop species primes viral activity in the rhizosphere through local adaptation. CONCLUSIONS: Overall, we reveal unprecedented spatial and temporal diversity in viral community composition and activity across root, rhizosphere soil, and bulk soil compartments. Our work demonstrates that the roles of soil viruses need greater consideration to exploit the rhizosphere microbiome for food security, food safety, and environmental sustainability. Video Abstract.

Quantitative Trait Locus Mapping of Resistance to Turnip Yellows Virus in Brassica rapa and Brassica oleracea and Introgression of These Resistances by Resynthesis Into Allotetraploid Plants for Deployment in Brassica napus.

Turnip yellows virus (TuYV) is aphid-transmitted and causes considerable yield losses in oilseed rape (OSR, Brassica napus, genome: AACC) and vegetable brassicas. Insecticide control of the aphid vector is limited due to insecticide resistance and the banning of the most effective active ingredients in the EU. There is only one source of TuYV resistance in current commercial OSR varieties, which has been mapped to a single dominant quantitative trait locus (QTL) on chromosome A04. We report the identification, characterisation, and mapping of TuYV resistance in the diploid progenitor species of OSR, Brassica rapa (genome: AA), and Brassica oleracea (genome: CC). Phenotyping of F1 populations, produced from within-species crosses between resistant and susceptible individuals, revealed the resistances were quantitative and partially dominant. QTL mapping of segregating backcross populations showed that the B. rapa resistance was controlled by at least two additive QTLs, one on chromosome A02 and the other on chromosome A06. Together, they explained 40.3% of the phenotypic variation. In B. oleracea, a single QTL on chromosome C05 explained 22.1% of the phenotypic variation. The TuYV resistance QTLs detected in this study are different from those in the extant commercial resistant varieties. To exploit these resistances, an allotetraploid (genome: AACC) plant line was resynthesised from the interspecific cross between the TuYV-resistant B. rapa and B. oleracea lines. Flow cytometry confirmed that plantlets regenerated from the interspecific cross had both A and C genomes and were mixoploid. To stabilise ploidy, a fertile plantlet was self-pollinated to produce seed that had the desired resynthesised, allotetraploid genome AACC. Phenotyping of the resynthesised plants confirmed their resistance to TuYV. Genotyping with resistance-linked markers identified during the mapping in the progenitors confirmed the presence of all TuYV resistance QTLs from B. rapa and B. oleracea. This is the first report of TuYV resistance mapped in the Brassica C genome and of an allotetraploid AACC line possessing dual resistance to TuYV originating from both of its progenitors. The introgression into OSR can now be accelerated, utilising marker-assisted selection, and this may reduce selection pressure for TuYV isolates that are able to overcome existing sources of resistance to TuYV.

The Evolutionary History of Wild, Domesticated, and Feral Brassica oleracea (Brassicaceae).

Understanding the evolutionary history of crops, including identifying wild relatives, helps to provide insight for conservation and crop breeding efforts. Cultivated Brassica oleracea has intrigued researchers for centuries due to its wide diversity in forms, which include cabbage, broccoli, cauliflower, kale, kohlrabi, and Brussels sprouts. Yet, the evolutionary history of this species remains understudied. With such different vegetables produced from a single species, B. oleracea is a model organism for understanding the power of artificial selection. Persistent challenges in the study of B. oleracea include conflicting hypotheses regarding domestication and the identity of the closest living wild relative. Using newly generated RNA-seq data for a diversity panel of 224 accessions, which represents 14 different B. oleracea crop types and nine potential wild progenitor species, we integrate phylogenetic and population genetic techniques with ecological niche modeling, archaeological, and literary evidence to examine relationships among cultivars and wild relatives to clarify the origin of this horticulturally important species. Our analyses point to the Aegean endemic B. cretica as the closest living relative of cultivated B. oleracea, supporting an origin of cultivation in the Eastern Mediterranean region. Additionally, we identify several feral lineages, suggesting that cultivated plants of this species can revert to a wild-like state with relative ease. By expanding our understanding of the evolutionary history in B. oleracea, these results contribute to a growing body of knowledge on crop domestication that will facilitate continued breeding efforts including adaptation to changing environmental conditions.

Identification of microbial signatures linked to oilseed rape yield decline at the landscape scale.

BACKGROUND: The plant microbiome plays a vital role in determining host health and productivity. However, we lack real-world comparative understanding of the factors which shape assembly of its diverse biota, and crucially relationships between microbiota composition and plant health. Here we investigated landscape scale rhizosphere microbial assembly processes in oilseed rape (OSR), the UK's third most cultivated crop by area and the world's third largest source of vegetable oil, which suffers from yield decline associated with the frequency it is grown in rotations. By including 37 conventional farmers' fields with varying OSR rotation frequencies, we present an innovative approach to identify microbial signatures characteristic of microbiomes which are beneficial and harmful to the host. RESULTS: We show that OSR yield decline is linked to rotation frequency in real-world agricultural systems. We demonstrate fundamental differences in the environmental and agronomic drivers of protist, bacterial and fungal communities between root, rhizosphere soil and bulk soil compartments. We further discovered that the assembly of fungi, but neither bacteria nor protists, was influenced by OSR rotation frequency. However, there were individual abundant bacterial OTUs that correlated with either yield or rotation frequency. A variety of fungal and protist pathogens were detected in roots and rhizosphere soil of OSR, and several increased relative abundance in root or rhizosphere compartments as OSR rotation frequency increased. Importantly, the relative abundance of the fungal pathogen Olpidium brassicae both increased with short rotations and was significantly associated with low yield. In contrast, the root endophyte Tetracladium spp. showed the reverse associations with both rotation frequency and yield to O. brassicae, suggesting that they are signatures of a microbiome which benefits the host. We also identified a variety of novel protist and fungal clades which are highly connected within the microbiome and could play a role in determining microbiome composition. CONCLUSIONS: We show that at the landscape scale, OSR crop yield is governed by interplay between complex communities of both pathogens and beneficial biota which is modulated by rotation frequency. Our comprehensive study has identified signatures of dysbiosis within the OSR microbiome, grown in real-world agricultural systems, which could be used in strategies to promote crop yield. Video abstract.

Cordycepin, a metabolite of Cordyceps militaris, reduces immune-related gene expression in insects.

Hypocrealean entomopathogenic fungi (EPF) (Sordariomycetes, Ascomycota) are natural regulators of insect populations in terrestrial environments. Their obligately-killing life-cycle means that there is likely to be strong selection pressure for traits that allow them to evade the effects of the host immune system. In this study, we quantified the effects of cordycepin (3'-deoxyadenosine), a secondary metabolite produced by Cordyceps militaris (Hypocreales, Cordycipitaceae), on insect susceptibility to EPF infection and on insect immune gene expression. Application of the immune stimulant curdlan (20 µg ml-1, linear beta-1,3-glucan, a constituent of fungal cell walls) to Drosophila melanogaster S2r+ cells resulted in a significant increase in the expression of the immune effector gene metchnikowin compared to a DMSO-only control, but there was no significant increase when curdlan was co-applied with 25 µg ml-1 cordycepin dissolved in DMSO. Injection of cordycepin into larvae of Galleria mellonella (Lepidoptera: Pyralidae) resulted in dose-dependent mortality (LC50 of cordycepin = 2.1 mg per insect 6 days after treatment). Incubating conidia of C. militaris and Beauveria bassiana (Hypocreales, Cordycipitaceae; an EPF that does not synthesize cordycepin) with 3.0 mg ml-1 cordycepin had no effect on the numbers of conidia germinating in vitro. Co-injection of G. mellonella with a low concentration of cordycepin (3.0 mg ml-1) plus 10 or 100 conidia per insect of C. militaris or B. bassiana caused a significant decrease in insect median survival time compared to injection with the EPF on their own. Analysis of predicted vs. observed mortalities indicated a synergistic interaction between cordycepin and the EPF. The injection of C. militaris and B. bassiana into G. mellonella resulted in increased expression of the insect immune effector genes lysozyme, IMPI and gallerimycin at 72 h post injection, but this did not occur when the EPF were co-injected with 3.0 mg ml-1 cordycepin. In addition, we observed increased expression of IMPI and lysozyme at 48 h after injection with C. militaris, B. bassiana and sham injection (indicating a wounding response), but this was also prevented by application of cordycepin. These results suggest that cordycepin has potential to act as a suppressor of the immune response during fungal infection of insect hosts.

Identification and QTL mapping of resistance to Turnip yellows virus (TuYV) in oilseed rape, Brassica napus.

KEY MESSAGE: Partially dominant resistance to Turnip yellows virus associated with one major QTL was identified in the natural allotetraploid oilseed rape cultivar Yudal. Turnip yellows virus (TuYV) is transmitted by the peach-potato aphid (Myzus persicae) and causes severe yield losses in commercial oilseed rape crops (Brassica napus). There is currently only one genetic resource for resistance to TuYV available in brassica, which was identified in the re-synthesised B. napus line 'R54'. In our study, 27 mostly homozygous B. napus accessions, either doubled-haploid (DH) or inbred lines, representing a diverse subset of the B. napus genepool, were screened for TuYV resistance/susceptibility. Partial resistance to TuYV was identified in the Korean spring oilseed rape, B. napus variety Yudal, whilst the dwarf French winter oilseed rape line Darmor-bzh was susceptible. QTL mapping using the established Darmor-bzh × Yudal DH mapping population (DYDH) revealed one major QTL explaining 36% and 18% of the phenotypic variation in two independent experiments. A DYDH line was crossed to Yudal, and reciprocal backcross (BC1) populations from the F1 with either the susceptible or resistant parent revealed the dominant inheritance of the TuYV resistance. The QTL on ChrA04 was verified in the segregating BC1 population. A second minor QTL on ChrC05 was identified in one of the two DYDH experiments, and it was not observed in the BC1 population. The TuYV resistance QTL in 'R54' is within the QTL interval on Chr A04 of Yudal; however, the markers co-segregating with the 'R54' resistance are not conserved in Yudal, suggesting an independent origin of the TuYV resistances. This is the first report of the QTL mapping of TuYV resistance in natural B. napus.

Assembly and characterisation of a unique onion diversity set identifies resistance to Fusarium basal rot and improved seedling vigour.

KEY MESSAGE: A unique, global onion diversity set was assembled, genotyped and phenotyped for beneficial traits. Accessions with strong basal rot resistance and increased seedling vigour were identified along with associated markers. Conserving biodiversity is critical for safeguarding future crop production. Onion (Allium cepa L.) is a globally important crop with a very large (16 Gb per 1C) genome which has not been sequenced. While onions are self-fertile, they suffer from severe inbreeding depression and as such are highly heterozygous as a result of out-crossing. Bulb formation is driven by daylength, and accessions are adapted to the local photoperiod. Onion seed is often directly sown in the field, and hence seedling establishment is a critical trait for production. Furthermore, onion yield losses regularly occur worldwide due to Fusarium basal rot caused by Fusarium oxysporum f. sp. cepae. A globally relevant onion diversity set, consisting of 10 half-sib families for each of 95 accessions, was assembled and genotyping carried out using 892 SNP markers. A moderate level of heterozygosity (30-35%) was observed, reflecting the outbreeding nature of the crop. Using inferred phylogenies, population structure and principal component analyses, most accessions grouped according to local daylength. A high level of intra-accession diversity was observed, but this was less than inter-accession diversity. Accessions with strong basal rot resistance and increased seedling vigour were identified along with associated markers, confirming the utility of the diversity set for discovering beneficial traits. The onion diversity set and associated trait data therefore provide a valuable resource for future germplasm selection and onion breeding.

Transcriptome and organellar sequencing highlights the complex origin and diversification of allotetraploid Brassica napus.

Brassica napus, an allotetraploid crop, is hypothesized to be a hybrid from unknown varieties of Brassica rapa and Brassica oleracea. Despite the economic importance of B. napus, much is unresolved regarding its phylogenomic relationships, genetic structure, and diversification. Here we conduct a comprehensive study among diverse accessions from 183 B. napus (including rapeseed, rutabaga, and Siberian kale), 112 B. rapa, and 62 B. oleracea and its wild relatives. Using RNA-seq of B. napus accessions, we define the genetic diversity and sub-genome variance of six genetic clusters. Nuclear and organellar phylogenies for B. napus and its progenitors reveal varying patterns of inheritance and post-formation introgression. We discern regions with signatures of selective sweeps and detect 8,187 differentially expressed genes with implications for B. napus diversification. This study highlights the complex origin and evolution of B. napus providing insights that can further facilitate B. napus breeding and germplasm preservation.

Towards new sources of resistance to the currant-lettuce aphid (Nasonovia ribisnigri).

Domesticated lettuce varieties encompass much morphological variation across a range of crop type groups, with large collections of cultivars and landrace accessions maintained in genebanks. Additional variation not captured during domestication, present in ancestral wild relatives, represents a potentially rich source of alleles that can deliver to sustainable crop production. However, these large collections are difficult and costly to screen for many agronomically important traits. In this paper, we describe the generation of a diversity collection of 96 lettuce and wild species accessions that are amenable to routine phenotypic analysis and their genotypic characterization with a panel of 682 newly developed expressed sequence tag (EST)-linked KASP™ single nucleotide polymorphism (SNP) markers that are anchored to the draft Lactuca sativa genome assembly. To exemplify the utility of these resources, we screened the collection for putative sources of resistance to currant-lettuce aphid (Nasonovia ribisnigri) and carried out association analyses to look for potential SNPs linked to resistance.

The pangenome of an agronomically important crop plant Brassica oleracea.

There is an increasing awareness that as a result of structural variation, a reference sequence representing a genome of a single individual is unable to capture all of the gene repertoire found in the species. A large number of genes affected by presence/absence and copy number variation suggest that it may contribute to phenotypic and agronomic trait diversity. Here we show by analysis of the Brassica oleracea pangenome that nearly 20% of genes are affected by presence/absence variation. Several genes displaying presence/absence variation are annotated with functions related to major agronomic traits, including disease resistance, flowering time, glucosinolate metabolism and vitamin biosynthesis.

Functional alleles of the flowering time regulator FRIGIDA in the Brassica oleracea genome.

BACKGROUND: Plants adopt different reproductive strategies as an adaptation to growth in a range of climates. In Arabidopsis thaliana FRIGIDA (FRI) confers a vernalization requirement and thus winter annual habit by increasing the expression of the MADS box transcriptional repressor FLOWERING LOCUS C (FLC). Variation at FRI plays a major role in A. thaliana life history strategy, as independent loss-of-function alleles that result in a rapid-cycling habit in different accessions, appear to have evolved many times. The aim of this study was to identify and characterize orthologues of FRI in Brassica oleracea. RESULTS: We describe the characterization of FRI from Brassica oleracea and identify the two B. oleracea FRI orthologues (BolC.FRI.a and BolC.FRI.b). These show extensive amino acid conservation in the central and C-terminal regions to FRI from other Brassicaceae, including A. thaliana, but have a diverged N-terminus. The genes map to two of the three regions of B. oleracea chromosomes syntenic to part of A. thaliana chromosome 5 suggesting that one of the FRI copies has been lost since the ancient triplication event that formed the B. oleracea genome. This genomic position is not syntenic with FRI in A. thaliana and comparative analysis revealed a recombination event within the A. thaliana FRI promoter. This relocated A. thaliana FRI to chromosome 4, very close to the nucleolar organizer region, leaving a fragment of FRI in the syntenic location on A. thaliana chromosome 5. Our data show this rearrangement occurred after the divergence from A. lyrata. We explored the allelic variation at BolC.FRI.a within cultivated B. oleracea germplasm and identified two major alleles, which appear equally functional both to each other and A. thaliana FRI, when expressed as fusions in A. thaliana. CONCLUSIONS: We identify the two Brassica oleracea FRI genes, one of which we show through A. thaliana complementation experiments is functional, and show their genomic location is not syntenic with A. thaliana FRI due to an ancient recombination event. This has complicated previous association analyses of FRI with variation in life history strategy in the Brassica genus.

Shoot calcium and magnesium concentrations differ between subtaxa, are highly heritable, and associate with potentially pleiotropic loci in Brassica oleracea.

Calcium (Ca) and magnesium (Mg) are the most abundant group II elements in both plants and animals. Genetic variation in shoot Ca and shoot Mg concentration (shoot Ca and Mg) in plants can be exploited to biofortify food crops and thereby increase dietary Ca and Mg intake for humans and livestock. We present a comprehensive analysis of within-species genetic variation for shoot Ca and Mg, demonstrating that shoot mineral concentration differs significantly between subtaxa (varietas). We established a structured diversity foundation set of 376 accessions to capture a high proportion of species-wide allelic diversity within domesticated Brassica oleracea, including representation of wild relatives (C genome, 1n = 9) from natural populations. These accessions and 74 modern F(1) hybrid cultivars were grown in glasshouse and field environments. Shoot Ca and Mg varied 2- and 2.3-fold, respectively, and was typically not inversely correlated with shoot biomass, within most subtaxa. The closely related capitata (cabbage) and sabauda (Savoy cabbage) subtaxa consistently had the highest mean shoot Ca and Mg. Shoot Ca and Mg in glasshouse-grown plants was highly correlated with data from the field. To understand and dissect the genetic basis of variation in shoot Ca and Mg, we studied homozygous lines from a segregating B. oleracea mapping population. Shoot Ca and Mg was highly heritable (up to 40%). Quantitative trait loci (QTL) for shoot Ca and Mg were detected on chromosomes C2, C6, C7, C8, and, in particular, C9, where QTL accounted for 14% to 55% of the total genetic variance. The presence of QTL on C9 was substantiated by scoring recurrent backcross substitution lines, derived from the same parents. This also greatly increased the map resolution, with strong evidence that a 4-cM region on C9 influences shoot Ca. This region corresponds to a 0.41-Mb region on Arabidopsis (Arabidopsis thaliana) chromosome 5 that includes 106 genes. There is also evidence that pleiotropic loci on C8 and C9 affect shoot Ca and Mg. Map-based cloning of these loci will reveal how shoot-level phenotypes relate to Ca(2+) and Mg(2+) uptake and homeostasis at the molecular level.

The reference genetic linkage map for the multinational Brassica rapa genome sequencing project.

We describe the construction of a reference genetic linkage map for the Brassica A genome, which will form the backbone for anchoring sequence contigs for the Multinational Brassica rapa Genome Sequencing Project. Seventy-eight doubled haploid lines derived from anther culture of the F(1) of a cross between two diverse Chinese cabbage (B. rapa ssp. pekinensis) inbred lines, 'Chiifu-401-42' (C) and 'Kenshin-402-43' (K) were used to construct the map. The map comprises a total of 556 markers, including 278 AFLP, 235 SSR, 25 RAPD and 18 ESTP, STS and CAPS markers. Ten linkage groups were identified and designated as R1-R10 through alignment and orientation using SSR markers in common with existing B. napus reference linkage maps. The total length of the linkage map was 1,182 cM with an average interval of 2.83 cM between adjacent loci. The length of linkage groups ranged from 81 to 161 cM for R04 and R06, respectively. The use of 235 SSR markers allowed us to align the A-genome chromosomes of B. napus with those of B. rapa ssp. pekinensis. The development of this map is vital to the integration of genome sequence and genetic information and will enable the international research community to share resources and data for the improvement of B. rapa and other cultivated Brassica species.

Integration of the cytogenetic and genetic linkage maps of Brassica oleracea.

We have assigned all nine linkage groups of a Brassica oleracea genetic map to each of the nine chromosomes of the karyotype derived from mitotic metaphase spreads of the B. oleracea var. alboglabra line A12DHd using FISH. The majority of probes were BACs, with A12DHd DNA inserts, which give clear, reliable FISH signals. We have added nine markers to the existing integrated linkage map, distributed over six linkage groups. BACs were definitively assigned to linkage map positions through development of locus-specific PCR assays. Integration of the cytogenetic and genetic linkage maps was achieved with 22 probes representing 19 loci. Four chromosomes (2, 4, 7, and 9) are in the same orientation as their respective linkage groups (O4, O7, O8, and O6) whereas four chromosomes (1, 3, 5, and 8) and linkage groups (O3, O9, O2, and O1) are in the opposite orientation. The remaining chromosome (6) is probably in the opposite orientation. The cytogenetic map is an important resource for locating probes with unknown genetic map positions and is also being used to analyze the relationships between genetic and cytogenetic maps.

Arabidopsis thaliana GATA factors: organisation, expression and DNA-binding characteristics.

Many light-responsive promoters contain GATA motifs and a number of nuclear proteins have been defined that interact with these elements. Type-IV zinc-finger proteins have been extensively characterised in animals and fungi and are referred to as GATA factors by virtue of their affinity for promoter elements containing this sequence. We previously identified cDNA sequences representing four Arabidopsis thaliana type-TV zinc-finger proteins. Here we define the organisation and expression of GATA-1, GATA-2, GATA-3 and GATA-4 as well as DNA-binding characteristics of their encoded proteins. Transcripts from all four genes can be detected in all tissues examined suggesting that they are not developmentally regulated at the level of transcription. In vitro binding experiments with Escherichia coli-derived recombinant proteins were performed using motifs previously defined as targets for nuclear GATA-binding proteins. These studies reveal differences in DNA binding specificity of GATA-1 as compared to the other three proteins. In vivo protein-DNA interactions monitored by yeast one-hybrid assays reveal different binding characteristics as compared to those defined with E. coli-derived recombinant protein. Trans-activation of gene expression by the four Arabidopsis proteins via some, but not all, DNA elements tested indicates that the Arabidopsis proteins can form functional interactions with previously defined promoter elements containing GATA motifs. We conclude that the Arabidopsis type-IV zinc-finger proteins may represent the previously defined family of nuclear GATA-binding proteins implicated in light-responsive transcription.