Cloning and functional validation of DsWRKY6 gene from Desmodium styracifolium.

The purpose of this study was to analyze the role of transcription factor in Desmodium styracifolium, proving that the DsWRKY6 transcription factor was related to the plant phenotypes of Desmodium styracifolium - cv. 'GuangYaoDa1' and it could be used in molecular-assisted breeding. 'GuangYaoDa1' was used as the material and its DNA was the template to clone DsWRKY6, the transgenic Arabidopsis thaliana line was constructed by agrobacterium tumefaciens­mediated transformation. Transgenic Arabidopsis thaliana was cultivated to study phenotype and physiological and biochemical indexes. Phenotypic observation showed that DsWRKY6 transgenic Arabidopsis thaliana had a faster growth rate while compared with the control group, they had longer lengths of main stem, lateral branches of cauline leaves, and root, but a lower number of cauline leaves and lateral branches of cauline leaves. And it also showed that their flowering and fruiting periods were advanced. The results of physiological and biochemical indexes showed that the relative expressions of DsWRKY6 increased and the abscisic acid content significantly increased in DsWRKY6 transgenic Arabidopsis thaliana compared with the control group. According to the above results, DsWRKY6 could regulate the advancing of flowering and fruiting periods caused by the improvement of abscisic acid content, and expression of the DsWRKY6 transcription factor might be the cause of the upright growth of 'GuangYaoDa1'.

Drought and heat stress: insights into tolerance mechanisms and breeding strategies for pigeonpea improvement.

MAIN CONCLUSION: Pigeonpea has potential to foster sustainable agriculture and resilience in evolving climate change; understanding bio-physiological and molecular mechanisms of heat and drought stress tolerance is imperative to developing resilience cultivars. Pigeonpea is an important legume crop that has potential resilience in the face of evolving climate scenarios. However, compared to other legumes, there has been limited research on abiotic stress tolerance in pigeonpea, particularly towards drought stress (DS) and heat stress (HS). To address this gap, this review delves into the genetic, physiological, and molecular mechanisms that govern pigeonpea's response to DS and HS. It emphasizes the need to understand how this crop combats these stresses and exhibits different types of tolerance and adaptation mechanisms through component traits. The current article provides a comprehensive overview of the complex interplay of factors contributing to the resilience of pigeonpea under adverse environmental conditions. Furthermore, the review synthesizes information on major breeding techniques, encompassing both conventional methods and modern molecular omics-assisted tools and techniques. It highlights the potential of genomics and phenomics tools and their pivotal role in enhancing adaptability and resilience in pigeonpea. Despite the progress made in genomics, phenomics and big data analytics, the complexity of drought and heat tolerance in pigeonpea necessitate continuous exploration at multi-omic levels. High-throughput phenotyping (HTP) is crucial for gaining insights into perplexed interactions among genotype, environment, and management practices (GxExM). Thus, integration of advanced technologies in breeding programs is critical for developing pigeonpea varieties that can withstand the challenges posed by climate change. This review is expected to serve as a valuable resource for researchers, providing a deeper understanding of the mechanisms underlying abiotic stress tolerance in pigeonpea and offering insights into modern breeding strategies that can contribute to the development of resilient varieties suited for changing environmental conditions.

Setting up Agrobacterium tumefaciens-mediated transformation of the tropical legume Aeschynomene evenia, a powerful tool for studying gene function in Nod Factor-independent symbiosis.

Most legumes are able to develop a root nodule symbiosis in association with proteobacteria collectively called rhizobia. Among them, the tropical species Aeschynomene evenia has the remarkable property of being nodulated by photosynthetic Rhizobia without the intervention of Nod Factors (NodF). Thereby, A. evenia has emerged as a working model for investigating the NodF-independent symbiosis. Despite the availability of numerous resources and tools to study the molecular basis of this atypical symbiosis, the lack of a transformation system based on Agrobacterium tumefaciens significantly limits the range of functional approaches. In this report, we present the development of a stable genetic transformation procedure for A. evenia. We first assessed its regeneration capability and found that a combination of two growth regulators, NAA (= Naphthalene Acetic Acid) and BAP (= 6-BenzylAminoPurine) allows the induction of budding calli from epicotyls, hypocotyls and cotyledons with a high efficiency in media containing 0,5 µM NAA (up to 100% of calli with continuous stem proliferation). To optimize the generation of transgenic lines, we employed A. tumefaciens strain EHA105 harboring a binary vector carrying the hygromycin resistance gene and the mCherry fluorescent marker. Epicotyls and hypocotyls were used as the starting material for this process. We have found that one growth medium containing a combination of NAA (0,5 µM) and BAP (2,2 µM) was sufficient to induce callogenesis and A. tumefaciens strain EHA105 was sufficiently virulent to yield a high number of transformed calli. This simple and efficient method constitutes a valuable tool that will greatly facilitate the functional studies in NodF-independent symbiosis.

Chromosome-level genome assembly and characterization of the Calophaca sinica genome.

Calophaca sinica is a rare plant endemic to northern China which belongs to the Fabaceae family and possesses rich nutritional value. To support the preservation of the genetic resources of this plant, we have successfully generated a high-quality genome of C. sinica (1.06 Gb). Notably, transposable elements (TEs) constituted ~73% of the genome, with long terminal repeat retrotransposons (LTR-RTs) dominating this group of elements (~54% of the genome). The average intron length of the C. sinica genome was noticeably longer than what has been observed for closely related species. The expansion of LTR-RTs and elongated introns emerged had the largest influence on the enlarged genome size of C. sinica in comparison to other Fabaceae species. The proliferation of TEs could be explained by certain modes of gene duplication, namely, whole genome duplication (WGD) and dispersed duplication (DSD). Gene family expansion, which was found to enhance genes associated with metabolism, genetic maintenance, and environmental stress resistance, was a result of transposed duplicated genes (TRD) and WGD. The presented genomic analysis sheds light on the genetic architecture of C. sinica, as well as provides a starting point for future evolutionary biology, ecology, and functional genomics studies centred around C. sinica and closely related species.

Global changes in gene expression during compatible and incompatible interactions of faba bean (Vicia faba L.) during Orobanche foetida parasitism.

Orobanche foetida Poiret is the main constraint facing faba bean crop in Tunisia. Indeed, in heavily infested fields with this parasitic plant, yield losses may reach 90%, and the recent estimation of the infested area is around 80,000 ha. Identifying genes involved in the Vicia faba/O. foetida interaction is crucial for the development of effective faba bean breeding programs. However, there is currently no available information on the transcriptome of faba bean responding to O. foetida parasitism. In this study, we employed RNA sequencing to explore the global gene expression changes associated with compatible and incompatible V. faba/O. foetida interactions. In this perspective, two faba bean varieties (susceptible and resistant) were examined at the root level across three stages of O. foetida development (Before Germination (BG), After Germination (AG) and Tubercule Stage (TS)). Our analyses presented an exploration of the transcriptomic profile, including comprehensive assessments of differential gene expression and Gene Ontology (GO) enrichment analyses. Specifically, we investigated key pathways revealing the complexity of molecular responses to O. foetida attack. In this study, we detected differential gene expression of pathways associated with secondary metabolites: flavonoids, auxin, thiamine, and jasmonic acid. To enhance our understanding of the global changes in V. faba response to O. foetida, we specifically examined WRKY genes known to play a role in plant host-parasitic plant interactions. Furthermore, considering the pivotal role of parasitic plant seed germination in this interaction, we investigated genes involved in the orobanchol biosynthesis pathway. Interestingly, we detected the gene expression of VuCYP722C homolog, coding for a key enzyme involved in orobanchol biosynthesis, exclusively in the susceptible host. Clearly, this study enriches our understanding of the V. faba/O. foetida interaction, shedding light on the main differences between susceptible and resistant faba bean varieties during O. foetida infestation at the gene expression level.

Metagenomics reveals the variations in functional metabolism associated with greenhouse gas emissions during legume-vegetable rotation process.

Legume-based rotation is commonly recognized for its mitigation efficiency of greenhouse gas (GHG) emissions. However, variations in GHG emission-associated metabolic functions during the legume-vegetable rotation process remain largely uncharacterized. Accordingly, a soybean-radish rotation field experiment was designed to clarify the responses of microbial communities and their GHG emission-associated functional metabolism through metagenomics. The results showed that the contents of soil organic carbon and total phosphorus significantly decreased during the soybean-radish process (P < 0.05), while soil total potassium content and bacterial richness and diversity significantly increased (P < 0.05). Moreover, the predominant bacterial phyla varied, with a decrease in the relative abundance of Proteobacteria and an increase in the relative abundance of Acidobacteria, Gemmatimonadetes, and Chloroflexi. Metagenomics clarified that bacterial carbohydrate metabolism substantially increased during the rotation process, whereas formaldehyde assimilation, methanogenesis, nitrification, and dissimilatory nitrate reduction decreased (P < 0.05). Specifically, the expression of phosphate acetyltransferase (functional methanogenesis gene, pta) and nitrate reductase gamma subunit (functional dissimilatory nitrate reduction gene, narI) was inhibited, indicating of low methane production and nitrogen metabolism. Additionally, the partial least squares path model revealed that the Shannon diversity index was negatively correlated with methane and nitrogen metabolism (P < 0.01), further demonstrating that the response of the soil bacterial microbiome responses are closely linked with GHG-associated metabolism during the soybean-radish rotation process. Collectively, our findings shed light on the responses of soil microbial communities to functional metabolism associated with GHG emissions and provide important insights to mitigate GHG emissions during the rotational cropping of legumes and vegetables.

AnDREB5.1, a A5 group DREB gene from desert shrub Ammopiptanthus nanus, confers osmotic and cold stress tolerances in transgenic tobacco.

The Dehydration-Responsive Element Binding (DREB) subfamily of transcription factors plays crucial roles in plant abiotic stress response. Ammopiptanthus nanus (A. nanus) is an eremophyte exhibiting remarkable tolerance to environmental stress and DREB proteins may contribute to its tolerance to water deficit and low-temperature stress. In the present study, an A. nanus DREB A5 group transcription factor gene, AnDREB5.1, was isolated and characterized in terms of structure and function in abiotic stress tolerance. AnDREB5.1 protein is distributed in the nucleus, possesses transactivation capacity, and is capable of binding to DRE core cis-acting element. The transcription of AnDREB5.1 was induced under osmotic and cold stress. Tobacco seedlings overexpressing AnDREB5.1 displayed higher tolerance to cold stress, osmotic stress, and oxidative stress compared to wild-type tobacco (WT). Under osmotic and cold stress, overexpression of AnDREB5.1 increased antioxidant enzyme activity in tobacco leaves, inhibiting excessive elevation of ROS levels. Transcriptome sequencing analysis showed that overexpression of AnDREB5.1 raised the tolerance of transgenic tobacco seedlings to abiotic stress by regulating multiple genes, including antioxidant enzymes, transcription factors, and stress-tolerant related functional genes like NtCOR413 and NtLEA14. This study provides new evidence for understanding the potential roles of the DREB A5 subgroup members in plants.

Transcriptomic and Physiological Analysis of the Effects of Exogenous Phloretin and Pterostilbene on Resistance Responses of Stylosanthes against Anthracnose.

Anthracnose caused by Colletotrichum gloeosporioides is a destructive disease of Stylosanthes (stylo). Combination treatment of phloretin and pterostilbene (PP) has been previously shown to effectively inhibit the conidial germination and mycelial growth of C. gloeosporioides in vitro. In this study, the effects of PP treatment on the growth of C. gloeosporioides in vivo and the biocontrol mechanisms were investigated. We found that exogenous PP treatment could limit the growth of C. gloeosporioides and alleviate the damage of anthracnose in stylo. Comparative transcriptome analysis revealed that 565 genes were up-regulated and 239 genes were down-regulated upon PP treatment during the infection by C. gloeosporioides. The differentially expressed genes were mainly related to oxidative stress and chloroplast organization. Further physiological analysis revealed that application of PP after C. gloeosporioides inoculation significantly reduced the accumulation of O2•- level and increased the accumulation of antioxidants (glutathione, ascorbic acid and flavonoids) as well as the enzyme activity of total antioxidant capacity, superoxide dismutase, catalase, glutathione reductase, peroxidase and ascorbate peroxidase. PP also reduced the decline of chlorophyll a + b and increased the content of carotenoid in response to C. gloeosporioides infection. These results suggest that PP treatment alleviates anthracnose by improving antioxidant capacity and reducing the damage of chloroplasts, providing insights into the biocontrol mechanisms of PP on the stylo against anthracnose.

Combining GWAS and comparative genomics to fine map candidate genes for days to flowering in mung bean.

BACKGROUND: Mung bean (Vigna radiata (L.) Wilczek), is an important pulse crop in the global south. Early flowering and maturation are advantageous traits for adaptation to northern and southern latitudes. This study investigates the genetic basis of the Days-to-Flowering trait (DTF) in mung bean, combining genome-wide association studies (GWAS) in mung bean and comparisons with orthologous genes involved with control of DTF responses in soybean (Glycine max (L) Merr) and Arabidopsis (Arabidopsis thaliana). RESULTS: The most significant associations for DTF were on mung bean chromosomes 1, 2, and 4. Only the SNPs on chromosomes 1 and 4 were heavily investigated using downstream analysis. The chromosome 1 DTF association is tightly linked with a cluster of locally duplicated FERONIA (FER) receptor-like protein kinase genes, and the SNP occurs within one of the FERONIA genes. In Arabidopsis, an orthologous FERONIA gene (AT3G51550), has been reported to regulate the expression of the FLOWERING LOCUS C (FLC). For the chromosome 4 DTF locus, the strongest candidates are Vradi04g00002773 and Vradi04g00002778, orthologous to the Arabidopsis PhyA and PIF3 genes, encoding phytochrome A (a photoreceptor protein sensitive to red to far-red light) and phytochrome-interacting factor 3, respectively. The soybean PhyA orthologs include the classical loci E3 and E4 (genes GmPhyA3, Glyma.19G224200, and GmPhyA2, Glyma.20G090000). The mung bean PhyA ortholog has been previously reported as a candidate for DTF in studies conducted in South Korea. CONCLUSION: The top two identified SNPs accounted for a significant proportion (~ 65%) of the phenotypic variability in mung bean DTF by the six significant SNPs (39.61%), with a broad-sense heritability of 0.93. The strong associations of DTF with genes that have orthologs with analogous functions in soybean and Arabidopsis provide strong circumstantial evidence that these genes are causal for this trait. The three reported loci and candidate genes provide useful targets for marker-assisted breeding in mung beans.

CRISPR/Cas9-mediated mutagenesis of phytoene desaturase in pigeonpea and groundnut.

The CRISPR/Cas9 technology, renowned for its ability to induce precise genetic alterations in various crop species, has encountered challenges in its application to grain legume crops such as pigeonpea and groundnut. Despite attempts at gene editing in groundnut, the low rates of transformation and editing have impeded its widespread adoption in producing genetically modified plants. This study seeks to establish an effective CRISPR/Cas9 system in pigeonpea and groundnut through Agrobacterium-mediated transformation, with a focus on targeting the phytoene desaturase (PDS) gene. The PDS gene is pivotal in carotenoid biosynthesis, and its disruption leads to albino phenotypes and dwarfism. Two constructs (one each for pigeonpea and groundnut) were developed for the PDS gene, and transformation was carried out using different explants (leaf petiolar tissue for pigeonpea and cotyledonary nodes for groundnut). By adjusting the composition of the growth media and refining Agrobacterium infection techniques, transformation efficiencies of 15.2% in pigeonpea and 20% in groundnut were achieved. Mutation in PDS resulted in albino phenotype, with editing efficiencies ranging from 4 to 6%. Sequence analysis uncovered a nucleotide deletion (A) in pigeonpea and an A insertion in groundnut, leading to a premature stop codon and, thereby, an albino phenotype. This research offers a significant foundation for the swift assessment and enhancement of CRISPR/Cas9-based genome editing technologies in legume crops.

Symbiotic efficiency of Rhizobium leguminosarum sv. trifolii strains originating from the subpolar and temperate climate regions.

Red clover (Trifolium pratense L.) is a forage legume cultivated worldwide. This plant is capable of establishing a nitrogen-fixing symbiosis with Rhizobium leguminosarum symbiovar trifolii strains. To date, no comparative analysis of the symbiotic properties and heterogeneity of T. pratense microsymbionts derived from two distinct geographic regions has been performed. In this study, the symbiotic properties of strains originating from the subpolar and temperate climate zones in a wide range of temperatures (10-25 °C) have been characterized. Our results indicate that all the studied T. pratense microsymbionts from two geographic regions were highly efficient in host plant nodulation and nitrogen fixation in a wide range of temperatures. However, some differences between the populations and between the strains within the individual population examined were observed. Based on the nodC and nifH sequences, the symbiotic diversity of the strains was estimated. In general, 13 alleles for nodC and for nifH were identified. Moreover, 21 and 61 polymorphic sites in the nodC and nifH sequences were found, respectively, indicating that the latter gene shows higher heterogeneity than the former one. Among the nodC and nifH alleles, three genotypes (I-III) were the most frequent, whereas the other alleles (IV-XIII) proved to be unique for the individual strains. Based on the nodC and nifH allele types, 20 nodC-nifH genotypes were identified. Among them, the most frequent were three genotypes marked as A (6 strains), B (5 strains), and C (3 strains). Type A was exclusively found in the temperate strains, whereas types B and C were identified in the subpolar strains. The remaining 17 genotypes were found in single strains. In conclusion, our data indicate that R. leguminosarum sv. trifolii strains derived from two climatic zones show a high diversity with respect to the symbiotic efficiency and heterogeneity. However, some of the R. leguminosarum sv. trifolii strains exhibit very good symbiotic potential in the wide range of the temperatures tested; hence, they may be used in the future for improvement of legume crop production.

The Glycine soja cytochrome P450 gene GsCYP82C4 confers alkaline tolerance by promoting reactive oxygen species scavenging.

Recent studies have demonstrated the crucial role of Cytochrome P450 enzymes (CYPs) in the production of secondary metabolites, phytohormones and antioxidants in plants. However, their functional characterization specifically under alkaline stress remains elusive. CYP82C4 was the key gene screened from a family of wild soybean CYPs in our previous studies. The aim of this present study was to clone the Glycine soja GsCYP82C4 gene and characterize its functions in Arabidopsis and Glycine max. The results showed that the GsCYP82C4 gene displayed a high expression in different plant tissues at mature stages compared to young stages. Further, higher temporal expression of the GsCYP82C4 gene was noted at 6, 12 and 24 h time points after alkali treatment in leaves compared to roots. In addition, overexpression of GsCYP82C4 improved alkaline stress tolerance in Arabidopsis via increased root lengths and fresh biomass and strengthened the antioxidant defense system via a reduction in superoxide radicals in transgenic lines compared to wild type (WT) and atcyp82c4 mutants. Further, the expression levels of stress-related marker genes were up-regulated in GsCYP82C4 OX lines under alkali stress. The functional analysis of GsCYP82C4 overexpression in soybean displayed better hairy root growth, increased fresh weight, higher antioxidant enzyme activities and reduced lipid peroxidation rates in OX lines compared to the soybean WT (K599) line. In total, our study displayed positive roles of GsCYP82C4 overexpression in both Arabidopsis and Glycine max to alleviate alkaline stress via altering expression abundance of stress responsive genes, stronger roots, higher antioxidant enzyme activities as well as reduced rates of lipid peroxidation and superoxide radicals.

Evolutionary history of an Irano-Turanian cushion-forming legume (Onobrychis cornuta).

The Irano-Turanian region is one of the largest floristic regions in the world and harbors a high percentage of endemics, including cushion-like and dwarf-shrubby taxa. Onobrychis cornuta is an important cushion-forming element of the subalpine/alpine flora of the Irano-Turanian floristic region. To specify the genetic diversity among the populations of this species (including individuals of O. elymaitica), we employed nrDNA ITS and two noncoding regions of plastid DNA (rpl32-trnL(UAG) and trnT(UGU)-trnL(UAA)). The most striking feature of O. cornuta assemblages was the unexpectedly high nucleotide diversity in both the nDNA and cpDNA dataset. In the analyses of nuclear and plastid regions, 25 ribotypes and 42 haplotypes were found among 77 and 59 accessions, respectively, from Iran, Turkey, and Afghanistan. Network analysis of the datasets demonstrated geographic differentiation within the species. Phylogenetic analyses of all dataset retrieved O. cornuta as a non-monophyletic species due to the inclusion of O. elymaitica, comprising four distinct lineages. In addition, our analyses showed cytonuclear discordance between both nuclear and plastid topologies regarding the position of some O. cornuta individuals. The underlying causes of this inconsistency remain unclear. However, we speculate that chloroplast capture, incomplete lineage sorting, and introgression were the main reasons for this event. Furthermore, molecular dating analysis indicated that O. cornuta originated in the early Pliocene (around 4.8 Mya) and started to diversify throughout the Pliocene and in particular the Pleistocene. Moreover, O. elymaitica was reduced to a subspecific rank within the species.

Deciphering Microbial Community and Nitrogen Fixation in the Legume Rhizosphere.

Nitrogen is the most limiting factor in crop production. Legumes establish a symbiotic relationship with rhizobia and enhance nitrogen fixation. We analyzed 1,624 rhizosphere 16S rRNA gene samples and 113 rhizosphere metagenomic samples from three typical legumes and three non-legumes. The rhizosphere microbial community of the legumes had low diversity and was enriched with nitrogen-cycling bacteria (Sphingomonadaceae, Xanthobacteraceae, Rhizobiaceae, and Bacillaceae). Furthermore, the rhizosphere microbiota of legumes exhibited a high abundance of nitrogen-fixing genes, reflecting a stronger nitrogen-fixing potential, and Streptomycetaceae and Nocardioidaceae were the predominant nitrogen-fixing bacteria. We also identified helper bacteria and confirmed through metadata analysis and a pot experiment that the synthesis of riboflavin by helper bacteria is the key factor in promoting nitrogen fixation. Our study emphasizes that the construction of synthetic communities of nitrogen-fixing bacteria and helper bacteria is crucial for the development of efficient nitrogen-fixing microbial fertilizers.

A genomic toolkit for winged bean Psophocarpus tetragonolobus.

A sustainable supply of plant protein is critical for future generations and needs to be achieved while reducing green house gas emissions from agriculture and increasing agricultural resilience in the face of climate volatility. Agricultural diversification with more nutrient-rich and stress tolerant crops could provide the solution. However, this is often hampered by the limited availability of genomic resources and the lack of understanding of the genetic structure of breeding germplasm and the inheritance of important traits. One such crop with potential is winged bean (Psophocarpus tetragonolobus), a high seed protein tropical legume which has been termed 'the soybean for the tropics'. Here, we present a chromosome level winged bean genome assembly, an investigation of the genetic diversity of 130 worldwide accessions, together with two linked genetic maps and a trait QTL analysis (and expression studies) for regions of the genome with desirable ideotype traits for breeding, namely architecture, protein content and phytonutrients.

Effects of Soil Rhizobia Abundance on Interactions between a Vector, Pathogen, and Legume Plant Host.

Soil rhizobia promote nitrogen fixation in legume hosts, maximizing their tolerance to different biotic stressors, plant biomass, crop growth, and yield. While the presence of soil rhizobia is considered beneficial for plants, few studies have assessed whether variation in rhizobia abundance affects the tolerance of legumes to stressors. To address this, we assessed the effects of variable soil rhizobia inoculum concentrations on interactions between a legume host (Pisum sativum), a vector insect (Acyrthosiphon pisum), and a virus (Pea enation mosaic virus, PEMV). We showed that increased rhizobia abundance reduces the inhibitory effects of PEMV on the nodule formation and root growth in 2-week-old plants. However, these trends were reversed in 4-week-old plants. Rhizobia abundance did not affect shoot growth or virus prevalence in 2- or 4-week-old plants. Our results show that rhizobia abundance may indirectly affect legume tolerance to a virus, but effects varied based on plant age. To assess the mechanisms that mediated interactions between rhizobia, plants, aphids, and PEMV, we measured the relative expression of gene transcripts related to plant defense signaling. Rhizobia concentrations did not strongly affect the expression of defense genes associated with phytohormone signaling. Our study shows that an abundance of soil rhizobia may impact a plant's ability to tolerate stressors such as vector-borne pathogens, as well as aid in developing sustainable pest and pathogen management systems for legume crops. More broadly, understanding how variable rhizobia concentrations can optimize legume-rhizobia symbiosis may enhance the productivity of legume crops.

Autotetraploid Induction of Three A-Genome Wild Peanut Species, Arachis cardenasii, A. correntina, and A. diogoi.

A-genome Arachis species (AA; 2n = 2x = 20) are commonly used as secondary germplasm sources in cultivated peanut breeding, Arachis hypogaea L. (AABB; 2n = 4x = 40), for the introgression of various biotic and abiotic stress resistance genes. Genome doubling is critical to overcoming the hybridization barrier of infertility that arises from ploidy-level differences between wild germplasm and cultivated peanuts. To develop improved genome doubling methods, four trials of various concentrations of the mitotic inhibitor treatments colchicine, oryzalin, and trifluralin were tested on the seedlings and seeds of three A-genome species, A. cardenasii, A. correntina, and A. diogoi. A total of 494 seeds/seedlings were treated in the present four trials, with trials 1 to 3 including different concentrations of the three chemical treatments on seedlings, and trial 4 focusing on the treatment period of 5 mM colchicine solution treatment of seeds. A small number of tetraploids were produced from the colchicine and oryzalin gel treatments of seedlings, but all these tetraploid seedlings reverted to diploid or mixoploid states within six months of treatment. In contrast, the 6-h colchicine solution treatment of seeds showed the highest tetraploid conversion rate (6-13% of total treated seeds or 25-40% of surviving seedlings), and the tetraploid plants were repeatedly tested as stable tetraploids. In addition, visibly and statistically larger leaves and flowers were produced by the tetraploid versions of these three species compared to their diploid versions. As a result, stable tetraploid plants of each A-genome species were produced, and a 5 mM colchicine seed treatment is recommended for A-genome and related wild Arachis species genome doubling.

Molecular phylogeny and divergence time of Harpalyce (Leguminosae, Papilionoideae), a lineage with amphitropical diversification in seasonally dry forests and savannas.

Our knowledge of the systematics of the papilionoid legume tribe Brongniartieae has greatly benefitted from recent advances in molecular phylogenetics. The tribe was initially described to include species marked by a strongly bilabiate calyx and an embryo with a straight radicle, but recent research has placed taxa from the distantly related core Sophoreae and Millettieae within it. Despite these advances, the most species-rich genera within the Brongniartieae are still not well studied, and their morphological and biogeographical evolution remains poorly understood. Comprising 35 species, Harpalyce is one of these poorly studied genera. In this study, we present a comprehensive, multi-locus molecular phylogeny of the Brongniartieae, with an increased sampling of Harpalyce, to investigate morphological and biogeographical evolution within the group. Our results confirm the monophyly of Harpalyce and indicate that peltate glandular trichomes and a strongly bilabiate calyx with a carinal lip and three fused lobes are synapomorphies for the genus, which is internally divided into three distinct ecologically and geographically divergent lineages, corresponding to the previously recognized sections. Our biogeographical reconstructions demonstrate that Brongniartieae originated in South America during the Eocene, with subsequent pulses of diversification in South America, Mesoamerica, and Australia. Harpalyce also originated in South America during the Miocene at around 20 Ma, with almost synchronous later diversification in South America and Mexico/Mesoamerica beginning 10 Ma, but mostly during the Pliocene. Migration of Harpalyce from South to North America was accompanied by a biome and ecological shift from savanna to seasonally dry forest.

Cytomolecular trends in Chamaecrista Moench (Caesalpinioideae, Leguminosae) diversification.

Chamaecrista is a Pantropical legume genus of the tribe Cassieae, which includes six other genera. In contrast to most of the other Cassieae genera, Chamaecrista shows significant variability in chromosome number (from 2n = 14 to 2n = 56), with small and morphologically similar chromosomes. Here, we performed a new cytomolecular analysis on chromosome number, genome size, and rDNA site distribution in a molecular phylogenetic perspective to interpret the karyotype trends of Chamaecrista and other two genera of Cassieae, seeking to understand their systematics and evolution. Our phylogenetic analysis revealed that Chamaecrista is monophyletic and can be divided into four major clades corresponding to the four sections of the genus. Chromosome numbers ranged from 2n = 14, 16 (section Chamaecrista) to 2n = 28 (sections Absus, Apoucouita, and Baseophyllum). The number of 5S and 35S rDNA sites varied between one and three pairs per karyotype, distributed on different chromosomes or in synteny, with no obvious phylogenetic significance. Our data allowed us to propose x = 7 as the basic chromosome number of Cassieae, which was changed by polyploidy generating x = 14 (sections Absus, Apoucouita, and Baseophyllum) and by ascending dysploidy to x = 8 (section Chamaecrista). The DNA content values supported this hypothesis, with the genomes of the putative tetraploids being larger than those of the putative diploids. We hypothesized that ascending dysploidy, polyploidy, and rDNA amplification/deamplification are the major events in the karyotypic diversification of Chamaecrista. The chromosomal marks characterized here may have cytotaxonomic potential in future studies.

From forest to savanna and back to forest: Evolutionary history of the genus Dimorphandra (Fabaceae).

The tree genus Dimorphandra (Fabaceae), which contains 26 species divided into three subgenera, was studied using DNA sequence data from six chloroplast genome regions (cpDNA) and the nuclear internal transcribed spacer (ITS). The analyses, which included Bayesian phylogenies and haplotype networks, ancestral area reconstructions, and ecological niche modeling, allowed for exploring the evolutionary history of Dimorphandra. Within the subgenus Phaneropsia, the cpDNA sequence data were more closely-related to species from the genus Mora, while the ITS sequence data displayed a closer phylogenetic relationship with the subgenus Pocillum. This incongruence may be due to incomplete lineage sorting associated with ancient polymorphisms. The Amazonian Dimophandra lineages were highly polymorphic and divergent, while those from the Cerrado and the Atlantic Forest had low levels of polymorphisms. The Amazon likely gave rise to the Dimophandra lineage that produced the Cerrado species, while a Cerrado lineage likely gave rise to the Atlantic Forest species. Habitat shifts were identified as a key factor in shaping the late evolutionary history of Dimorphandra.