An ex vitro hairy root system from petioles of detached soybean leaves for in planta screening of target genes and CRISPR strategies associated with nematode bioassays.

MAIN CONCLUSION: The ex vitro hairy root system from petioles of detached soybean leaves allows the functional validation of genes using classical transgenesis and CRISPR strategies (e.g., sgRNA validation, gene activation) associated with nematode bioassays. Agrobacterium rhizogenes-mediated root transformation has been widely used in soybean for the functional validation of target genes in classical transgenesis and single-guide RNA (sgRNA) in CRISPR-based technologies. Initial data showed that in vitro hairy root induction from soybean cotyledons and hypocotyls were not the most suitable strategies for simultaneous performing genetic studies and nematode bioassays. Therefore, an ex vitro hairy root system was developed for in planta screening of target molecules during soybean parasitism by root-knot nematodes (RKNs). Applying this method, hairy roots were successfully induced by A. rhizogenes from petioles of detached soybean leaves. The soybean GmPR10 and GmGST genes were then constitutively overexpressed in both soybean hairy roots and tobacco plants, showing a reduction in the number of Meloidogyne incognita-induced galls of up to 41% and 39%, respectively. In addition, this system was evaluated for upregulation of the endogenous GmExpA and GmExpLB genes by CRISPR/dCas9, showing high levels of gene activation and reductions in gall number of up to 58.7% and 67.4%, respectively. Furthermore, morphological and histological analyses of the galls were successfully performed. These collective data validate the ex vitro hairy root system for screening target genes, using classical overexpression and CRISPR approaches, directly in soybean in a simple manner and associated with nematode bioassays. This system can also be used in other root pathosystems for analyses of gene function and studies of parasite interactions with plants, as well as for other purposes such as studies of root biology and promoter characterization.

Ectopic expression of an expansin-like B gene from wild Arachis enhances tolerance to both abiotic and biotic stresses.

Plant expansins are structural cell wall-loosening proteins implicated in several developmental processes and responses to environmental constraints and pathogen infection. To date, there is limited information about the biological function of expansins-like B (EXLBs), one of the smallest and less-studied subfamilies of plant expansins. In the present study, we conducted a functional analysis of the wild Arachis AdEXLB8 gene in transgenic tobacco (Nicotiana tabacum) plants to clarify its putative role in mediating defense responses to abiotic and biotic stresses. First, its cell wall localization was confirmed in plants expressing an AdEXLB8:eGFP fusion protein, while nanomechanical assays indicated cell wall reorganization and reassembly due to AdEXLB8 overexpression without compromising the phenotype. We further demonstrated that AdEXLB8 increased tolerance not only to isolated abiotic (drought) and biotic (Sclerotinia sclerotiorum and Meloidogyne incognita) stresses but also to their combination. The jasmonate and abscisic acid signaling pathways were clearly favored in transgenic plants, showing an activated antioxidative defense system. In addition to modifications in the biomechanical properties of the cell wall, we propose that AdEXLB8 overexpression interferes with phytohormone dynamics leading to a defense primed state, which culminates in plant defense responses against isolated and combined abiotic and biotic stresses.

Segmental allopolyploidy in action: Increasing diversity through polyploid hybridization and homoeologous recombination.

PREMISE OF THE STUDY: The genetic bottleneck of polyploid formation can be mitigated by multiple origins, gene flow, and recombination among different lineages. In crop plants with limited origins, efforts to increase genetic diversity have limitations. Here we used lineage recombination to increase genetic diversity in peanut, an allotetraploid likely of single origin, by crossing with a novel allopolyploid genotype and selecting improved lines. METHODS: Single backcross progeny from cultivated peanut × wild species-derived allotetraploid cross were studied over successive generations. Using genetic assumptions that encompass segmental allotetraploidy, we used single nucleotide polymorphisms and whole-genome sequence data to infer genome structures. KEY RESULTS: Selected lines, despite a high proportion of wild alleles, are agronomically adapted, productive, and with improved disease resistances. Wild alleles mostly substituted homologous segments of the peanut genome. Regions of dispersed wild alleles, characteristic of gene conversion, also occurred. However, wild chromosome segments sometimes replaced cultivated peanut's homeologous subgenome; A. ipaënsis B sometimes replaced A. hypogaea A subgenome (~0.6%), and A. duranensis replaced A. hypogaea B subgenome segments (~2%). Furthermore, some subgenome regions historically lost in cultivated peanut were "recovered" by wild chromosome segments (effectively reversing the "polyploid ratchet"). These processes resulted in lines with new genome structure variations. CONCLUSIONS: Genetic diversity was introduced by wild allele introgression, and by introducing new genome structure variations. These results highlight the special possibilities of segmental allotetraploidy and of using lineage recombination to increase genetic diversity in peanut, likely mirroring what occurs in natural segmental allopolyploids with multiple origins.

The genome structure of Arachis hypogaea (Linnaeus, 1753) and an induced Arachis allotetraploid revealed by molecular cytogenetics.

Peanut, Arachis hypogaea (Linnaeus, 1753) is an allotetraploid cultivated plant with two subgenomes derived from the hybridization between two diploid wild species, A. duranensis (Krapovickas & W. C. Gregory, 1994) and A. ipaensis (Krapovickas & W. C. Gregory, 1994), followed by spontaneous chromosomal duplication. To understand genome changes following polyploidy, the chromosomes of A. hypogaea, IpaDur1, an induced allotetraploid (A. ipaensis × A. duranensis)4x and the diploid progenitor species were cytogenetically compared. The karyotypes of the allotetraploids share the number and general morphology of chromosomes; DAPI+ bands pattern and number of 5S rDNA loci. However, one 5S rDNA locus presents a heteromorphic FISH signal in both allotetraploids, relative to corresponding progenitor. Whilst for A. hypogaea the number of 45S rDNA loci was equivalent to the sum of those present in the diploid species, in IpaDur1, two loci have not been detected. Overall distribution of repetitive DNA sequences was similar in both allotetraploids, although A. hypogaea had additional CMA3+ bands and few slight differences in the LTR-retrotransposons distribution compared to IpaDur1. GISH showed that the chromosomes of both allotetraploids had preferential hybridization to their corresponding diploid genomes. Nevertheless, at least one pair of IpaDur1 chromosomes had a clear mosaic hybridization pattern indicating recombination between the subgenomes, clear evidence that the genome of IpaDur1 shows some instability comparing to the genome of A. hypogaea that shows no mosaic of subgenomes, although both allotetraploids derive from the same progenitor species. For some reasons, the chromosome structure of A. hypogaea is inherently more stable, or, it has been at least, partially stabilized through genetic changes and selection.

Phenotypic effects of allotetraploidization of wild Arachis and their implications for peanut domestication.

PREMISE OF THE STUDY: Several species of Arachis have been cultivated for their edible seeds, historically and to the present day. The diploid species that have a history of cultivation show relatively small signatures of domestication. In contrast, the tetraploid species A. hypogaea evolved into highly domesticated forms and became a major world crop, the cultivated peanut. It seems likely that allotetraploidization (hybridity and/or tetraploidization) in some way enhanced attractiveness for cultivation. Here we investigate this using six different hybridization and tetraploidization events, from distinct Arachis diploid species, including one event derived from the same wild species that originated peanut. METHODS: Twenty-six anatomical, morphological, and physiological traits were examined in the induced allotetraploid plants and compared with their wild diploid parents. KEY RESULTS: Nineteen traits were transgressive (showed strong response to hybridization and chromosome duplication): allotetraploids had larger leaves, stomata and epidermal cells than did their diploid parents. In addition, allotetraploids produced more photosynthetic pigments. These traits have the same trend across the different hybrid combinations, suggesting that the changes are more likely due to ploidy rather than hybridity. In contrast, seed dimensions and seed mass did not significantly change in response to hybridization or tetraploidization. CONCLUSIONS: We suggest that the original allotetraploid that gave rise to cultivated peanut may have been attractive because of an increase in plant size, different transpiration characteristics, higher photosynthetic capacity, or other characteristics, but contrary to accepted knowledge, increased seed size was unlikely to have been important in the initial domestication.

Genetic Mapping of Resistance to Meloidogyne arenaria in Arachis stenosperma: A New Source of Nematode Resistance for Peanut.

Root-knot nematodes (RKN; Meloidogyne sp.) are a major threat to crops in tropical and subtropical regions worldwide. The use of resistant crop varieties is the preferred method of control because nematicides are expensive, and hazardous to humans and the environment. Peanut (Arachis hypogaea) is infected by four species of RKN, the most damaging being M. arenaria, and commercial cultivars rely on a single source of resistance. In this study, we genetically characterize RKN resistance of the wild Arachis species A. stenosperma using a population of 93 recombinant inbred lines developed from a cross between A. duranensis and A. stenosperma. Four quantitative trait loci (QTL) located on linkage groups 02, 04, and 09 strongly influenced nematode root galling and egg production. Drought-related, domestication and agronomically relevant traits were also evaluated, revealing several QTL. Using the newly available Arachis genome sequence, easy-to-use KASP (kompetitive allele specific PCR) markers linked to the newly identified RKN resistance loci were developed and validated in a tetraploid context. Therefore, we consider that A. stenosperma has high potential as a new source of RKN resistance in peanut breeding programs.

Arachis batizocoi: a study of its relationship to cultivated peanut (A. hypogaea) and its potential for introgression of wild genes into the peanut crop using induced allotetraploids.

BACKGROUND AND AIMS: Arachis batizocoi is a wild relative of cultivated peanut (A. hypogaea), an allotetraploid with an AABB genome. Arachis batizocoi was once considered the ancestral donor of the peanut B genome, but cytogenetics and DNA phylogenies have indicated a new genome classification, 'K'. These observations seem inconsistent with genetic studies and breeding that have shown that A. batizocoi can behave as a B genome. METHODS: The genetic behaviour, genome composition and phylogenetic position of A. batizocoi were studied using controlled hybridizations, induced tetraploidy, whole-genome in situ fluorescent hybridization (GISH) and molecular phylogenetics. KEY RESULTS: Sterile diploid hybrids containing AK genomes were obtained using A. batizocoi and the A genome species A. duranensis, A. stenosperma, A. correntina or A. villosa. From these, three types of AAKK allotetraploids were obtained, each in multiple independent polyploidy events. Induced allotetraploids were vigorous and fertile, and were hybridized to A. hypogaea to produce F1 hybrids. Even with the same parental combination, fertility of these F1 hybrids varied greatly, suggesting the influence of stochastic genetic or epigenetic events. Interestingly, hybrids with A. hypogaea ssp. hypogaea were significantly more fertile than those with the subspecies fastigiata. GISH in cultivated × induced allotetraploids hybrids (harbouring AABK genomes) and a molecular phylogeny using 16 intron sequences showed that the K genome is distinct, but more closely related to the B than to the A genome. CONCLUSIONS: The K genome of A. batizocoi is more related to B than to the A genome, but is distinct. As such, when incorporated in an induced allotetraploid (AAKK) it can behave as a B genome in crosses with peanut. However, the fertility of hybrids and their progeny depends upon the compatibility of the A genome interactions. The genetic distinctness of A. batizocoi makes it an important source of allelic diversity in itself, especially in crosses involving A. hypogaea ssp. hypogaea.

Suicide risk and alcohol and drug abuse in outpatients with HIV infection and Chagas disease

Objective: To evaluate psychiatric comorbidities in outpatients receiving care for HIV and Chagas disease at Instituto de Pesquisa Clínica Evandro Chagas (IPEC), Fundação Oswaldo Cruz (Fiocruz), Rio de Janeiro, Brazil. Methods: Cross-sectional study with a consecutive sample of 125 patients referred to an outpatient psychiatric clinic from February to December 2010. The Mini International Neuropsychiatric Interview (MINI) was used. Factors associated with more frequent mental disorders were estimated by odds ratios (OR) with 95% confidence intervals (95%CI) by multiple logistic regression. Results: Seventy-six (60.8%) patients with HIV, 40 (32%) patients with Chagas disease, and nine (7.2%) patients with human T-lymphotropic virus were interviewed. The majority were women (64%), with up to 8 years of formal education (56%), and unemployed (81.6%). The median age was 49 years. Suicide risk (n=71) (56%), agoraphobia (n=65) (52%), major depressive episode (n=56) (44.8%), and alcohol/drug abuse (n=43) (34.4%) predominated, the latter being directly associated with lower family income (OR = 2.64; 95%CI 1.03-6.75) and HIV infection (OR = 5.24; 95%CI 1.56-17.61). Suicide risk was associated with non-white skin color (OR = 2.21; 95%CI 1.03-4.75), unemployment (OR = 2.72; 95%CI 1.01-7.34), and diagnosis of major depression (OR = 3.34; 95%CI 1.54-7.44). Conclusion: Measures targeting adverse socioeconomic conditions and psychiatric and psychological monitoring and care should be encouraged in this population, considering the association with abuse of alcohol/other psychoactive drugs and suicide risk. .

The use of SNP markers for linkage mapping in diploid and tetraploid peanuts.

Single nucleotide polymorphic markers (SNPs) are attractive for use in genetic mapping and marker-assisted breeding because they can be scored in parallel assays at favorable costs. However, scoring SNP markers in polyploid plants like the peanut is problematic because of interfering signal generated from the DNA bases that are homeologous to those being assayed. The present study used a previously constructed 1536 GoldenGate SNP assay developed using SNPs identified between two A. duranensis accessions. In this study, the performance of this assay was tested on two RIL mapping populations, one diploid (A. duranensis × A. stenosperma) and one tetraploid [A. hypogaea cv. Runner IAC 886 × synthetic tetraploid (A. ipaënsis × A. duranensis)(4×)]. The scoring was performed using the software GenomeStudio version 2011.1. For the diploid, polymorphic markers provided excellent genotyping scores with default software parameters. In the tetraploid, as expected, most of the polymorphic markers provided signal intensity plots that were distorted compared to diploid patterns and that were incorrectly scored using default parameters. However, these scorings were easily corrected using the GenomeStudio software. The degree of distortion was highly variable. Of the polymorphic markers, approximately 10% showed no distortion at all behaving as expected for single-dose markers, and another 30% showed low distortion and could be considered high-quality. The genotyped markers were incorporated into diploid and tetraploid genetic maps of Arachis and, in the latter case, were located almost entirely on A genome linkage groups.

Matita, a new retroelement from peanut: characterization and evolutionary context in the light of the Arachis A-B genome divergence.

Cultivated peanut is an allotetraploid with an AB-genome. In order to learn more of the genomic structure of peanut, we characterized and studied the evolution of a retrotransposon originally isolated from a resistance gene analog (RGA)-containing bacterial artificial chromosome (BAC) clone. It is a moderate copy number Ty1-copia retrotransposon from the Bianca lineage and we named it Matita. Fluorescent in situ hybridization (FISH) experiments showed that Matita is mainly located on the distal regions of chromosome arms and is of approximately equal frequency on both A- and B-chromosomes. Its chromosome-specific hybridization pattern facilitates the identification of individual chromosomes, a useful cytogenetic tool considering that chromosomes in peanut are mostly metacentric and of similar size. Phylogenetic analysis of Matita elements, molecular dating of transposition events, and an estimation of the evolutionary divergence of the most probable A- and B-donor species suggest that Matita underwent its last major burst of transposition activity at around the same time of the A- and B-genome divergence about 3.5 million years ago. By probing BAC libraries with overgos probes for Matita, resistance gene analogues, and single- or low-copy genes, it was demonstrated that Matita is not randomly distributed in the genome but exhibits a significant tendency of being more abundant near resistance gene homologues than near single-copy genes. The described work is a further step towards broadening the knowledge on genomic and chromosomal structure of peanut and on its evolution.

A linkage map for the B-genome of Arachis (Fabaceae) and its synteny to the A-genome.

BACKGROUND: Arachis hypogaea (peanut) is an important crop worldwide, being mostly used for edible oil production, direct consumption and animal feed. Cultivated peanut is an allotetraploid species with two different genome components, A and B. Genetic linkage maps can greatly assist molecular breeding and genomic studies. However, the development of linkage maps for A. hypogaea is difficult because it has very low levels of polymorphism. This can be overcome by the utilization of wild species of Arachis, which present the A- and B-genomes in the diploid state, and show high levels of genetic variability. RESULTS: In this work, we constructed a B-genome linkage map, which will complement the previously published map for the A-genome of Arachis, and produced an entire framework for the tetraploid genome. This map is based on an F2 population of 93 individuals obtained from the cross between the diploid A. ipaënsis (K30076) and the closely related A. magna (K30097), the former species being the most probable B genome donor to cultivated peanut. In spite of being classified as different species, the parents showed high crossability and relatively low polymorphism (22.3%), compared to other interspecific crosses. The map has 10 linkage groups, with 149 loci spanning a total map distance of 1,294 cM. The microsatellite markers utilized, developed for other Arachis species, showed high transferability (81.7%). Segregation distortion was 21.5%. This B-genome map was compared to the A-genome map using 51 common markers, revealing a high degree of synteny between both genomes. CONCLUSION: The development of genetic maps for Arachis diploid wild species with A- and B-genomes effectively provides a genetic map for the tetraploid cultivated peanut in two separate diploid components and is a significant advance towards the construction of a transferable reference map for Arachis. Additionally, we were able to identify affinities of some Arachis linkage groups with Medicago truncatula, which will allow the transfer of information from the nearly-complete genome sequences of this model legume to the peanut crop.

BAC libraries construction from the ancestral diploid genomes of the allotetraploid cultivated peanut.

BACKGROUND: Cultivated peanut, Arachis hypogaea is an allotetraploid of recent origin, with an AABB genome. In common with many other polyploids, it seems that a severe genetic bottle-neck was imposed at the species origin, via hybridisation of two wild species and spontaneous chromosome duplication. Therefore, the study of the genome of peanut is hampered both by the crop's low genetic diversity and its polyploidy. In contrast to cultivated peanut, most wild Arachis species are diploid with high genetic diversity. The study of diploid Arachis genomes is therefore attractive, both to simplify the construction of genetic and physical maps, and for the isolation and characterization of wild alleles. The most probable wild ancestors of cultivated peanut are A. duranensis and A. ipaënsis with genome types AA and BB respectively. RESULTS: We constructed and characterized two large-insert libraries in Bacterial Artificial Chromosome (BAC) vector, one for each of the diploid ancestral species. The libraries (AA and BB) are respectively c. 7.4 and c. 5.3 genome equivalents with low organelle contamination and average insert sizes of 110 and 100 kb. Both libraries were used for the isolation of clones containing genetically mapped legume anchor markers (single copy genes), and resistance gene analogues. CONCLUSION: These diploid BAC libraries are important tools for the isolation of wild alleles conferring resistances to biotic stresses, comparisons of orthologous regions of the AA and BB genomes with each other and with other legume species, and will facilitate the construction of a physical map.