Genome assembly and analysis of Lactuca virosa: implications for lettuce breeding.

Lettuce (Lactuca sativa L.) is a leafy vegetable crop with ongoing breeding efforts related to quality, resilience, and innovative production systems. To breed resilient and resistant lettuce in the future, valuable genetic variation found in close relatives could be further exploited. Lactuca virosa (2x = 2n = 18), a wild relative assigned to the tertiary lettuce gene pool, has a much larger genome (3.7 Gbp) than Lactuca sativa (2.5 Gbp). It has been used in interspecific crosses and is a donor to modern crisphead lettuce cultivars. Here, we present a de novo reference assembly of L. virosa with high continuity and complete gene space. This assembly facilitated comparisons to the genome of L. sativa and to that of the wild species L. saligna, a representative of the secondary lettuce gene pool. To assess the diversity in gene content, we classified the genes of the 3 Lactuca species as core, accessory, and unique. In addition, we identified 3 interspecific chromosomal inversions compared to L. sativa, which each may cause recombination suppression and thus hamper future introgression breeding. Using 3-way comparisons in both reference-based and reference-free manners, we show that the proliferation of long-terminal repeat elements has driven the genome expansion of L. virosa. Further, we performed a genome-wide comparison of immune genes, nucleotide-binding leucine-rich repeat, and receptor-like kinases among Lactuca spp. and indicated the evolutionary patterns and mechanisms behind their expansions. These genome analyses greatly facilitate the understanding of genetic variation in L. virosa, which is beneficial for the breeding of improved lettuce varieties.

Genetic Mapping of Genotype-by-Ploidy Effects in Arabidopsis thaliana.

Plants can express different phenotypic responses following polyploidization, but ploidy-dependent phenotypic variation has so far not been assigned to specific genetic factors. To map such effects, segregating populations at different ploidy levels are required. The availability of an efficient haploid inducer line in Arabidopsis thaliana allows for the rapid development of large populations of segregating haploid offspring. Because Arabidopsis haploids can be self-fertilised to give rise to homozygous doubled haploids, the same genotypes can be phenotyped at both the haploid and diploid ploidy level. Here, we compared the phenotypes of recombinant haploid and diploid offspring derived from a cross between two late flowering accessions to map genotype × ploidy (G × P) interactions. Ploidy-specific quantitative trait loci (QTLs) were detected at both ploidy levels. This implies that mapping power will increase when phenotypic measurements of monoploids are included in QTL analyses. A multi-trait analysis further revealed pleiotropic effects for a number of the ploidy-specific QTLs as well as opposite effects at different ploidy levels for general QTLs. Taken together, we provide evidence of genetic variation between different Arabidopsis accessions being causal for dissimilarities in phenotypic responses to altered ploidy levels, revealing a G × P effect. Additionally, by investigating a population derived from late flowering accessions, we revealed a major vernalisation-specific QTL for variation in flowering time, countering the historical bias of research in early flowering accessions.

The genome of Lactuca saligna, a wild relative of lettuce, provides insight into non-host resistance to the downy mildew Bremia lactucae.

Lactuca saligna L. is a wild relative of cultivated lettuce (Lactuca sativa L.), with which it is partially interfertile. Hybrid progeny suffer from hybrid incompatibility (HI), resulting in reduced fertility and distorted transmission ratios. Lactuca saligna displays broad-spectrum resistance against lettuce downy mildew caused by Bremia lactucae Regel and is considered a non-host species. This phenomenon of resistance in L. saligna is called non-host resistance (NHR). One possible mechanism behind this NHR is through the plant-pathogen interaction triggered by pathogen recognition receptors, including nucleotide-binding leucine-rich repeat (NLR) proteins and receptor-like kinases (RLKs). We report a chromosome-level genome assembly of L. saligna (accession CGN05327), leading to the identification of two large paracentric inversions (>50 Mb) between L. saligna and L. sativa. Genome-wide searches delineated the major resistance clusters as regions enriched in NLRs and RLKs. Three of the enriched regions co-locate with previously identified NHR intervals. RNA-seq analysis of Bremia-infected lettuce identified several differentially expressed RLKs in NHR regions. Three tandem wall-associated kinase-encoding genes (WAKs) in the NHR8 interval display particularly high expression changes at an early stage of infection. We propose RLKs as strong candidates for determinants of the NHR phenotype of L. saligna.

The Gynandropsis gynandra genome provides insights into whole-genome duplications and the evolution of C4 photosynthesis in Cleomaceae.

Gynandropsis gynandra (Cleomaceae) is a cosmopolitan leafy vegetable and medicinal plant, which has also been used as a model to study C4 photosynthesis due to its evolutionary proximity to C3 Arabidopsis (Arabidopsis thaliana). Here, we present the genome sequence of G. gynandra, anchored onto 17 main pseudomolecules with a total length of 740 Mb, an N50 of 42 Mb and 30,933 well-supported gene models. The G. gynandra genome and previously released genomes of C3 relatives in the Cleomaceae and Brassicaceae make an excellent model for studying the role of genome evolution in the transition from C3 to C4 photosynthesis. Our analyses revealed that G. gynandra and its C3 relative Tarenaya hassleriana shared a whole-genome duplication event (Gg-α), then an addition of a third genome (Th-α, +1×) took place in T. hassleriana but not in G. gynandra. Analysis of syntenic copy number of C4 photosynthesis-related gene families indicates that G. gynandra generally retained more duplicated copies of these genes than C3T. hassleriana, and also that the G. gynandra C4 genes might have been under positive selection pressure. Both whole-genome and single-gene duplication were found to contribute to the expansion of the aforementioned gene families in G. gynandra. Collectively, this study enhances our understanding of the polyploidy history, gene duplication and retention, as well as their impact on the evolution of C4 photosynthesis in Cleomaceae.

The genome sequence of Hirschfeldia incana, a new Brassicaceae model to improve photosynthetic light-use efficiency.

Photosynthesis is a key process in sustaining plant and human life. Improving the photosynthetic capacity of agricultural crops is an attractive means to increase their yields. While the core mechanisms of photosynthesis are highly conserved in C3 plants, these mechanisms are very flexible, allowing considerable diversity in photosynthetic properties. Among this diversity is the maintenance of high photosynthetic light-use efficiency at high irradiance as identified in a small number of exceptional C3 species. Hirschfeldia incana, a member of the Brassicaceae family, is such an exceptional species, and because it is easy to grow, it is an excellent model for studying the genetic and physiological basis of this trait. Here, we present a reference genome of H. incana and confirm its high photosynthetic light-use efficiency. While H. incana has the highest photosynthetic rates found so far in the Brassicaceae, the light-saturated assimilation rates of closely related Brassica rapa and Brassica nigra are also high. The H. incana genome has extensively diversified from that of B. rapa and B. nigra through large chromosomal rearrangements, species-specific transposon activity, and differential retention of duplicated genes. Duplicated genes in H. incana, B. rapa, and B. nigra that are involved in photosynthesis and/or photoprotection show a positive correlation between copy number and gene expression, providing leads into the mechanisms underlying the high photosynthetic efficiency of these species. Our work demonstrates that the H. incana genome serves as a valuable resource for studying the evolution of high photosynthetic light-use efficiency and enhancing photosynthetic rates in crop species.

Bracon brevicornis Genome Showcases the Potential of Linked-Read Sequencing in Identifying a Putative Complementary Sex Determiner Gene.

Bracon brevicornis is an ectoparasitoid of a wide range of larval-stage Lepidopterans, including several pests of important crops, such as the corn borer, Ostrinia nubilalis. It is also one of the earliest documented cases of complementary sex determination in Hymenoptera. Here, we present the linked-read-based genome of B. brevicornis, complete with an ab initio-derived annotation and protein comparisons with fellow braconids, Fopius arisanus and Diachasma alloeum. We demonstrate the potential of linked-read assemblies in exploring regions of heterozygosity and search for structural and homology-derived evidence of the complementary sex determiner gene (csd).

Reciprocal cybrids reveal how organellar genomes affect plant phenotypes.

Assessment of the impact of variation in chloroplast and mitochondrial DNA (collectively termed the plasmotype) on plant phenotypes is challenging due to the difficulty in separating their effect from nuclear-derived variation (the nucleotype). Haploid-inducer lines can be used as efficient plasmotype donors to generate new plasmotype-nucleotype combinations (cybrids)1. We generated a panel comprising all possible cybrids of seven Arabidopsis thaliana accessions and extensively phenotyped these lines for 1,859 phenotypes under both stable and fluctuating conditions. We show that natural variation in the plasmotype results in both additive and epistatic effects across all phenotypic categories. Plasmotypes that induce more additive phenotypic changes also cause more epistatic effects, suggesting a possible common basis for both additive and epistatic effects. On average, epistatic interactions explained twice as much of the variance in phenotypes as additive plasmotype effects. The impact of plasmotypic variation was also more pronounced under fluctuating and stressful environmental conditions. Thus, the phenotypic impact of variation in plasmotypes is the outcome of multi-level nucleotype-plasmotype-environment interactions and, as such, the plasmotype is likely to serve as a reservoir of variation that is predominantly exposed under certain conditions. The production of cybrids using haploid inducers is a rapid and precise method for assessment of the phenotypic effects of natural variation in organellar genomes. It will facilitate efficient screening of unique nucleotype-plasmotype combinations to both improve our understanding of natural variation in these combinations and identify favourable combinations to enhance plant performance.

Assessment of heterosis in two Arabidopsis thaliana common-reference mapping populations.

Hybrid vigour, or heterosis, has been of tremendous importance in agriculture for the improvement of both crops and livestock. Notwithstanding large efforts to study the phenomenon of heterosis in the last decades, the identification of common molecular mechanisms underlying hybrid vigour remain rare. Here, we conducted a systematic survey of the degree of heterosis in Arabidopsis thaliana hybrids. For this purpose, two overlapping Arabidopsis hybrid populations were generated by crossing a large collection of naturally occurring accessions to two common reference lines. In these Arabidopsis hybrid populations the range of heterosis for several developmental and yield related traits was examined, and the relationship between them was studied. The traits under study were projected leaf area at 17 days after sowing, flowering time, height of the main inflorescence, number of side branches from the main stem or from the rosette base, total seed yield, seed weight, seed size and the estimated number of seeds per plant. Predominantly positive heterosis was observed for leaf area and height of the main inflorescence, whereas mainly negative heterosis was observed for rosette branching. For the other traits both positive and negative heterosis was observed in roughly equal amounts. For flowering time and seed size only low levels of heterosis were detected. In general the observed heterosis levels were highly trait specific. Furthermore, no correlation was observed between heterosis levels and the genetic distance between the parental lines. Since all selected lines were a part of the Arabidopsis genome wide association (GWA) mapping panel, a genetic mapping approach was applied to identify possible regions harbouring genetic factors causal for heterosis, with separate calculations for additive and dominance effects. Our study showed that the genetic mechanisms underlying heterosis were highly trait specific in our hybrid populations and greatly depended on the genetic background, confirming the elusive character of heterosis.

Genome-Wide Association Mapping Reveals That Specific and Pleiotropic Regulatory Mechanisms Fine-Tune Central Metabolism and Growth in Arabidopsis.

Central metabolism is a coordinated network that is regulated at multiple levels by resource availability and by environmental and developmental cues. Its genetic architecture has been investigated by mapping metabolite quantitative trait loci (QTL). A more direct approach is to identify enzyme activity QTL, which distinguishes between cis-QTL in structural genes encoding enzymes and regulatory trans-QTL. Using genome-wide association studies, we mapped QTL for 24 enzyme activities, nine metabolites, three structural components, and biomass in Arabidopsis thaliana We detected strong cis-QTL for five enzyme activities. A cis-QTL for UDP-glucose pyrophosphorylase activity in the UGP1 promoter is maintained through balancing selection. Variation in acid invertase activity reflects multiple evolutionary events in the promoter and coding region of VAC-INVcis-QTL were also detected for ADP-glucose pyrophosphorylase, fumarase, and phosphoglucose isomerase activity. We detected many trans-QTL, including transcription factors, E3 ligases, protein targeting components, and protein kinases, and validated some by knockout analysis. trans-QTL are more frequent but tend to have smaller individual effects than cis-QTL. We detected many colocalized QTL, including a multitrait QTL on chromosome 4 that affects six enzyme activities, three metabolites, protein, and biomass. These traits are coordinately modified by different ACCELERATED CELL DEATH6 alleles, revealing a trade-off between metabolism and defense against biotic stress.

Natural variation in life history strategy of Arabidopsis thaliana determines stress responses to drought and insects of different feeding guilds.

Plants are sessile organisms and, consequently, are exposed to a plethora of stresses in their local habitat. As a result, different populations of a species are subject to different selection pressures leading to adaptation to local conditions and intraspecific divergence. The annual brassicaceous plant Arabidopsis thaliana is an attractive model for ecologists and evolutionary biologists due to the availability of a large collection of resequenced natural accessions. Accessions of A. thaliana display one of two different life cycle strategies: summer and winter annuals. We exposed a collection of 308 European Arabidopsis accessions, that have been genotyped for 250K SNPs, to a range of stresses: one abiotic stress (drought), four biotic stresses (Pieris rapae caterpillars, Plutella xylostella caterpillars, Frankliniella occidentalis thrips and Myzus persicae aphids) and two combined stresses (drought plus P. rapae and Botrytis cinerea fungus plus P. rapae). We identified heritable genetic variation for responses to the different stresses, estimated by narrow-sense heritability. We found that accessions displaying different life cycle strategies differ in their response to stresses. Winter annuals are more resistant to drought, aphids and thrips and summer annuals are more resistant to P. rapae and P. xylostella caterpillars. Summer annuals are also more resistant to the combined stresses of drought plus P. rapae and infection by the fungus Botryris cinerea plus herbivory by P. rapae. Adaptation to drought displayed a longitudinal gradient. Finally, trade-offs were recorded between the response to drought and responses to herbivory by caterpillars of the specialist herbivore P. rapae.

Genome-Wide Association Mapping of Fertility Reduction upon Heat Stress Reveals Developmental Stage-Specific QTLs in Arabidopsis thaliana.

For crops that are grown for their fruits or seeds, elevated temperatures that occur during flowering and seed or fruit set have a stronger effect on yield than high temperatures during the vegetative stage. Even short-term exposure to heat can have a large impact on yield. In this study, we used Arabidopsis thaliana to study the effect of short-term heat exposure on flower and seed development. The impact of a single hot day (35°C) was determined in more than 250 natural accessions by measuring the lengths of the siliques along the main inflorescence. Two sensitive developmental stages were identified, one before anthesis, during male and female meiosis, and one after anthesis, during fertilization and early embryo development. In addition, we observed a correlation between flowering time and heat tolerance. Genome-wide association mapping revealed four quantitative trait loci (QTLs) strongly associated with the heat response. These QTLs were developmental stage specific, as different QTLs were detected before and after anthesis. For a number of QTLs, T-DNA insertion knockout lines could validate assigned candidate genes. Our findings show that the regulation of complex traits can be highly dependent on the developmental timing.

Trypanocidal Activity of Smallanthus sonchifolius: Identification of Active Sesquiterpene Lactones by Bioassay-Guided Fractionation.

In order to find novel plant-derived biologically active compounds against Trypanosoma cruzi, we isolated, from the organic extract of Smallanthus sonchifolius, the sesquiterpene lactones enhydrin (1), uvedalin (2), and polymatin B (3) by bioassay-guided fractionation technique. These compounds showed a significant trypanocidal activity against the epimastigote forms of the parasite with IC50 values of 0.84 µ M (1), 1.09 µ M (2), and 4.90 µ M (3). After a 24 h treatment with 10 µ g/mL of enhydrin or uvedalin, parasites were not able to recover their replication rate. Compounds 1 and 2 showed IC50 values of 33.4 µ M and 25.0 µ M against T. cruzi trypomastigotes, while polymatin B was not active. When the three compounds were tested against the intracellular forms of T. cruzi, they were able to inhibit the amastigote replication with IC50 of 5.17 µ M, 3.34 µ M, and 9.02 µ M for 1, 2, and 3, respectively. The cytotoxicity of the compounds was evaluated in Vero cells obtaining CC50 values of 46.5 µ M (1), 46.8 µ M (2), and 147.3 µ M (3) and the selectivity index calculated. According to these results, enhydrin and uvedalin might have potentials as agents against Chagas disease and could serve as lead molecules to develop new drugs.

Toxoplasma gondii infection blocks the development of allergic airway inflammation in BALB/c mice.

There is a link between increased allergy and a reduction of some infections in western countries. Epidemiological data also show that respiratory allergy is less frequent in people exposed to orofaecal and foodborne microbes such as Toxoplasma gondii. Infection with T. gondii induces a strong cell-mediated immunity with a highly polarized T helper type 1 (Th1) response in early stages of infection. Using a well-known murine model of allergic lung inflammation, we sought to investigate whether T. gondii infection could modulate the susceptibility to develop respiratory allergies. Both acute and chronic infection with T. gondii before allergic sensitization resulted in a diminished allergic inflammation, as shown by a decrease in bronchoalveolar lavage (BAL) eosinophilia, mononuclear and eosinophil cell infiltration around airways and vessels and goblet cell hyperplasia. Low allergen-specific immunoglobulin (Ig)E and IgG1 and high levels of allergen-specific IgG2a serum antibodies were detected. A decreased interleukin (IL)-4 and IL-5 production by lymph node cells was observed, while no antigen-specific interferon-gamma increase was detected. Higher levels of the regulatory cytokine IL-10 were found in BAL from infected mice. These results show that both acute and chronic parasite infection substantially blocked development of airway inflammation in adult BALB/c mice. Our results support the hypothesis that T. gondii infection contributes to protection against allergy in humans.

Elicitation of specific, Th1-biased immune response precludes skeletal muscle damage in cruzipain-vaccinated mice.

Cruzipain (Cz), the major cystein proteinase of Trypanosoma cruzi, is able to induce protective immunity against parasite challenge. However, some concern has arisen regarding its potential to elicit pathogenic autoimmune reactivity. To determine whether the adverse myopathic effects of Cz-based immunization could be prevented, we evaluated the co-administration of Cz with different adjuvants. Mice were immunized with Cz adjuvantized by alum (Cz+alum), oligodeoxynucleotides containing CpG motifs (Cz+ODN-CpG) or Freund's preparation (Cz+CFA). Cz triggered a vigorous specific humoral response, irrespective of the adjuvant used. Alum mainly drove response towards Th2 phenotype, characterized by specific IgG1 antibodies and IL-10 induction, whereas Cz+ODN-CpG mice exhibited Th1-dominant immunity, with antibodies of the IgG2a isotype and enhanced IFN-gamma production. Histological examination of cardiac tissue demonstrated lesions in Cz+CFA but not in Cz+alum nor Cz+ODN-CpG immunized animals, suggesting that CFA is critical for Cz-mediated injury. Analysis of skeletal muscle revealed that mice receiving Cz+CFA exhibited disrupted and hyalinized myofibers, whereas [Cz+alum]-immunized animals showed hyalinization, architecture modifications and small inflammatory foci. Conversely, no abnormalities were observed in the striated muscle from the Cz+ODN-CpG group. Hence, generation of specific immune response skewed towards Th1, as that recorded for the ODN-CpG adjuvant, may preclude triggering of Cz-mediated muscle tissue damage.

Characterization of human infection by Leishmania spp. in the Northwest of Argentina: immune response, double infection with Trypanosoma cruzi and species of Leishmania involved.

The aims of this study were to characterize human American tegumentary leishmaniasis, which includes cutaneous, mucocutaneous and mucosal leishmaniasis, in Northwest Argentina, to determine the prevalence of double infection with Trypanosoma cruzi and to identify the species of Leishmania in this area. Most of the 330 leishmaniasis patients presented cutaneous ulcers (96.1%), 2.4% mucocutaneous and 1.5% the mucosal form ('espundia'). The aetiological agents, determined by isoenzyme electrophoresis, were identified as Leishmania (Viannia) braziliensis in 16 out of 20 isolates and in the remaining 4 as Leishmania (Leishimania) amazonensis, the first ever-documented in Argentina. Sera analysed by ELISA and IFA using complex antigen from both T. cruzi and L. braziliensis showed a very high percentage of positives (66.3-78.2%). When antigens for specific diagnosis of Chagas' disease were used, 40.9% of the leishmaniasis patients were also found to be infected by T. cruzi. These results indicate that the strong immune response against T. cruzi gave no protection to Leishmania, in spite of the serological cross-reaction between these parasites. In addition, we showed that more than 40% of the patients would be misdiagnosed as chagasic if complex antigens, as epimastigotes or soluble fraction from epimastigotes, were used in IFA or ELISA. This is of paramount importance not only because patients' treatment would be associated to misdiagnosis but the fact that in many countries in Central and South America, a positive test for Chagas' disease means a rejection for those seeking employment.

Clinical status and parasitic infection in a Wichí Aboriginal community in Salta, Argentina.

In a study, carried out in 2000, of the clinical and parasitological status of a Wichi Aboriginal community living in the suburbs of Tartagal, northern Salta, Argentina, 154 individuals were screened for parasitic infections. Ninety-five faecal samples were also obtained from the same population. Ninety-three percent of the subjects were positive for 1 or more of the parasites investigated by direct test and 70.5% of them had parasitic superinfection. The most frequent helminths were Strongyloides stercoralis (50.5%) and hookworm (47.4%). We found low reinfection rates and a long reinfection period after treatment and provision of safe water and sanitation. Serum reactivity of these patients was analysed by enzyme-linked immunosorbent assay and indirect immunofluorescent assay and 22.1% of them had anti-Toxocara antibodies, 16.2% were positive for a complex antigen of Leishmania braziliensis, 29.9% were positive for a complex Trypanosoma cruzi antigen, and 17.5% were positive for a specific Trypanosoma cruzi antigen, Ag 163B6/cruzipain.

Role of placental alkaline phosphatase in the interaction between human placental trophoblast and Trypanosoma cruzi.

Congenital Chagas disease, due to the intracellular parasite Trypanosoma cruzi, is associated with premature labor, miscarriage, and placentitis. Human enzyme placental alkaline phosphatase (PLAP) (EC 3.1.3.1.) is membrane-anchored through glycosylphosphatidylinositol (GPI). PLAP is present in plasma in late pregnancy, 36 to 40 weeks; there are lower levels in maternal Chagas disease. Infants born to such mothers may have congenital Chagas disease. Human placental villi (PV) were treated with phospholipase-C (PL-C) and then cultured with T. cruzi to determine the effect of the parasites on PLAP activity as an in vitro model. There is less PLAP activity after treatment by PL-C and during culture with T. cruzi. Pretreatment of PV with PL-C before culture with T. cruzi yielded essentially normal specific activity of PLAP and prevented or greatly reduced infective penetration of villi by parasites. The results are consistent with a pathogenetic role for placental alkaline phosphatase in congenital Chagas disease. Receptor activation of membrane attachment to PLAP may be a device used by T. cruzi to enable parasite invasion of human trophoblast.

Polymerase chain reaction reveals Trypanosoma cruzi infection suspected by serology in cutaneous and mucocutaneous leishmaniasis patients.

The existence of patients suffering a double infection caused by Trypanosoma cruzi and Leishmania spp. has been suggested by several authors. Since the conventional serological tests now available for the diagnosis of Chagas' disease lack specificity due to the cross-reactivity between these two parasites, a serological confirmation of a T. cruzi infection cannot be made unless specific antigens are used. An enzyme linked immunosorbent assay (ELISA) to detect antibodies against a specific T. cruzi antigen, named Ag163B6, and immunoblotting using T. cruzi epimastigotes, are non-conventional serological techniques that could be employed for specific diagnosis of Chagas' disease. Using these two methods 34 cutaneous or mucocutaneous leishmaniasis patients were classified into two groups: (A) patients with serological evidence of T. cruzi infection, i.e. those who tested positive in at least one assay (18/34); and (B) patients with no serological evidence of T. cruzi infection, i.e. those who were negative for both assays (16/34). Taking into account the difficulties of xenodiagnosis and its low sensitivity (less than 50%) for a direct diagnosis in the chronic period of the disease, we used polymerase chain reaction (PCR) to confirm a T. cruzi infection in those leishmaniasis patients who presented positive results with the non-conventional serological techniques. Of the 18 patients with serological evidence of T. cruzi infection, 17 gave positive results when genomic DNA primers were used. Using minicircle primers, 15/18 of that group were positive. Nevertheless, all the patients suspected of being double infected were positive in at least one PCR test. Just one patient with no serological evidence of T. cruzi infection gave a positive PCR result when amplifying the minicircle sequence. The proof of the existence of a T. cruzi infection by PCR in leishmaniasis patients suspected to be chagasic when non-conventional serology was used, strongly supports the use of the specific Ag163B6 and immunoblotting with epimastigotes as specific serological diagnostic tools to determine a T. cruzi infection.