Transcriptomic response to nitrogen availability reveals signatures of adaptive plasticity during tetraploid wheat domestication.

The domestication of crops, coupled with agroecosystem development, is associated with major environmental changes and provides an ideal model of phenotypic plasticity. Here, we examined 32 genotypes of three tetraploid wheat (Triticum turgidum L.) subspecies, wild emmer, emmer and durum wheat, which are representative of the key stages in the domestication of tetraploid wheat. We developed a pipeline that integrates RNA-Seq data and population genomics to assess gene expression plasticity and identify selection signatures under diverse nitrogen availability conditions. Our analysis revealed differing gene expression responses to nitrogen availability across primary (wild emmer to emmer) and secondary (emmer to durum wheat) domestication. Notably, nitrogen triggered the expression of twice as many genes in durum wheat compared to that in emmer and wild emmer. Unique selection signatures were identified at each stage: primary domestication mainly influenced genes related to biotic interactions, whereas secondary domestication affected genes related to amino acid metabolism, in particular lysine. Selection signatures were found in differentially expressed genes, notably those associated with nitrogen metabolism, such as the gene encoding glutamate dehydrogenase. Overall, our study highlights the pivotal role of nitrogen availability in the domestication and adaptive responses of a major food crop, with varying effects across different traits and growth conditions.

CRISPR-Cas9-based repeat depletion for high-throughput genotyping of complex plant genomes.

High-throughput genotyping enables the large-scale analysis of genetic diversity in population genomics and genome-wide association studies that combine the genotypic and phenotypic characterization of large collections of accessions. Sequencing-based approaches for genotyping are progressively replacing traditional genotyping methods because of the lower ascertainment bias. However, genome-wide genotyping based on sequencing becomes expensive in species with large genomes and a high proportion of repetitive DNA. Here we describe the use of CRISPR-Cas9 technology to deplete repetitive elements in the 3.76-Gb genome of lentil (Lens culinaris), 84% consisting of repeats, thus concentrating the sequencing data on coding and regulatory regions (single-copy regions). We designed a custom set of 566,766 gRNAs targeting 2.9 Gbp of repeats and excluding repetitive regions overlapping annotated genes and putative regulatory elements based on ATAC-seq data. The novel depletion method removed ∼40% of reads mapping to repeats, increasing those mapping to single-copy regions by ∼2.6-fold. When analyzing 25 million fragments, this repeat-to-single-copy shift in the sequencing data increased the number of genotyped bases of ∼10-fold compared to nondepleted libraries. In the same condition, we were also able to identify ∼12-fold more genetic variants in the single-copy regions and increased the genotyping accuracy by rescuing thousands of heterozygous variants that otherwise would be missed because of low coverage. The method performed similarly regardless of the multiplexing level, type of library or genotypes, including different cultivars and a closely related species (L. orientalis). Our results showed that CRISPR-Cas9-driven repeat depletion focuses sequencing data on single-copy regions, thus improving high-density and genome-wide genotyping in large and repetitive genomes.

A comprehensive metabolomics and lipidomics atlas for the legumes common bean, chickpea, lentil and lupin.

Legumes represent an important component of human and livestock diets; they are rich in macro- and micronutrients such as proteins, dietary fibers and polyunsaturated fatty acids. Whilst several health-promoting and anti-nutritional properties have been associated with grain content, in-depth metabolomics characterization of major legume species remains elusive. In this article, we used both gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) to assess the metabolic diversity in the five legume species commonly grown in Europe, including common bean (Phaseolus vulgaris), chickpea (Cicer arietinum), lentil (Lens culinaris), white lupin (Lupinus albus) and pearl lupin (Lupinus mutabilis), at the tissue level. We were able to detect and quantify over 3400 metabolites covering major nutritional and anti-nutritional compounds. Specifically, the metabolomics atlas includes 224 derivatized metabolites, 2283 specialized metabolites and 923 lipids. The data generated here will serve the community as a basis for future integration to metabolomics-assisted crop breeding and facilitate metabolite-based genome-wide association studies to dissect the genetic and biochemical bases of metabolism in legume species.

Genotype Combinations Drive Variability in the Microbiome Configuration of the Rhizosphere of Maize/Bean Intercropping System.

In an intercropping system, the interplay between cereals and legumes, which is strongly driven by the complementarity of below-ground structures and their interactions with the soil microbiome, raises a fundamental query: Can different genotypes alter the configuration of the rhizosphere microbial communities? To address this issue, we conducted a field study, probing the effects of intercropping and diverse maize (Zea mays L.) and bean (Phaseolus vulgaris L., Phaseolus coccineus L.) genotype combinations. Through amplicon sequencing of bacterial 16S rRNA genes from rhizosphere samples, our results unveil that the intercropping condition alters the rhizosphere bacterial communities, but that the degree of this impact is substantially affected by specific genotype combinations. Overall, intercropping allows the recruitment of exclusive bacterial species and enhances community complexity. Nevertheless, combinations of maize and bean genotypes determine two distinct groups characterized by higher or lower bacterial community diversity and complexity, which are influenced by the specific bean line associated. Moreover, intercropped maize lines exhibit varying propensities in recruiting bacterial members with more responsive lines showing preferential interactions with specific microorganisms. Our study conclusively shows that genotype has an impact on the rhizosphere microbiome and that a careful selection of genotype combinations for both species involved is essential to achieve compatibility optimization in intercropping.

CRISPR/Cas9-Mediated Enrichment Coupled to Nanopore Sequencing Provides a Valuable Tool for the Precise Reconstruction of Large Genomic Target Regions.

Complete and accurate identification of genetic variants associated with specific phenotypes can be challenging when there is a high level of genomic divergence between individuals in a study and the corresponding reference genome. We have applied the Cas9-mediated enrichment coupled to nanopore sequencing to perform a targeted de novo assembly and accurately reconstruct a genomic region of interest. This approach was used to reconstruct a 250-kbp target region on chromosome 5 of the common bean genome (Phaseolus vulgaris) associated with the shattering phenotype. Comparing a non-shattering cultivar (Midas) with the reference genome revealed many single-nucleotide variants and structural variants in this region. We cut five 50-kbp tiled sub-regions of Midas genomic DNA using Cas9, followed by sequencing on a MinION device and de novo assembly, generating a single contig spanning the whole 250-kbp region. This assembly increased the number of Illumina reads mapping to genes in the region, improving their genotypability for downstream analysis. The Cas9 tiling approach for target enrichment and sequencing is a valuable alternative to whole-genome sequencing for the assembly of ultra-long regions of interest, improving the accuracy of downstream genotype-phenotype association analysis.

The INCREASE project: Intelligent Collections of food-legume genetic resources for European agrofood systems.

Food legumes are crucial for all agriculture-related societal challenges, including climate change mitigation, agrobiodiversity conservation, sustainable agriculture, food security and human health. The transition to plant-based diets, largely based on food legumes, could present major opportunities for adaptation and mitigation, generating significant co-benefits for human health. The characterization, maintenance and exploitation of food-legume genetic resources, to date largely unexploited, form the core development of both sustainable agriculture and a healthy food system. INCREASE will implement, on chickpea (Cicer arietinum), common bean (Phaseolus vulgaris), lentil (Lens culinaris) and lupin (Lupinus albus and L. mutabilis), a new approach to conserve, manage and characterize genetic resources. Intelligent Collections, consisting of nested core collections composed of single-seed descent-purified accessions (i.e., inbred lines), will be developed, exploiting germplasm available both from genebanks and on-farm and subjected to different levels of genotypic and phenotypic characterization. Phenotyping and gene discovery activities will meet, via a participatory approach, the needs of various actors, including breeders, scientists, farmers and agri-food and non-food industries, exploiting also the power of massive metabolomics and transcriptomics and of artificial intelligence and smart tools. Moreover, INCREASE will test, with a citizen science experiment, an innovative system of conservation and use of genetic resources based on a decentralized approach for data management and dynamic conservation. By promoting the use of food legumes, improving their quality, adaptation and yield and boosting the competitiveness of the agriculture and food sector, the INCREASE strategy will have a major impact on economy and society and represents a case study of integrative and participatory approaches towards conservation and exploitation of crop genetic resources.

Pod indehiscence in common bean is associated with the fine regulation of PvMYB26.

In legumes, pod shattering occurs when mature pods dehisce along the sutures, and detachment of the valves promotes seed dispersal. In Phaseolus vulgaris (L)., the major locus qPD5.1-Pv for pod indehiscence was identified recently. We developed a BC4/F4 introgression line population and narrowed the major locus down to a 22.5 kb region. Here, gene expression and a parallel histological analysis of dehiscent and indehiscent pods identified an AtMYB26 orthologue as the best candidate for loss of pod shattering, on a genomic region ~11 kb downstream of the highest associated peak. Based on mapping and expression data, we propose early and fine up-regulation of PvMYB26 in dehiscent pods. Detailed histological analysis establishes that pod indehiscence is associated with the lack of a functional abscission layer in the ventral sheath, and that the key anatomical modifications associated with pod shattering in common bean occur early during pod development. We finally propose that loss of pod shattering in legumes resulted from histological convergent evolution and that it is the result of selection at orthologous loci.

Genomic dissection of pod shattering in common bean: mutations at non-orthologous loci at the basis of convergent phenotypic evolution under domestication of leguminous species.

The complete or partial loss of shattering ability occurred independently during the domestication of several crops. Therefore, the study of this trait can provide an understanding of the link between phenotypic and molecular convergent evolution. The genetic dissection of 'pod shattering' in Phaseolus vulgaris is achieved here using a population of introgression lines and next-generation sequencing techniques. The 'occurrence' of the indehiscent phenotype (indehiscent versus dehiscent) depends on a major locus on chromosome 5. Furthermore, at least two additional genes are associated with the 'level' of shattering (number of shattering pods per plant: low versus high) and the 'mode' of shattering (non-twisting versus twisting pods), with all of these loci contributing to the phenotype by epistatic interactions. Comparative mapping indicates that the major gene identified on common bean chromosome 5 corresponds to one of the four quantitative trait loci for pod shattering in Vigna unguiculata. None of the loci identified comprised genes that are homologs of the known shattering genes in Glycine max. Therefore, although convergent domestication can be determined by mutations at orthologous loci, this was only partially true for P. vulgaris and V. unguiculata, which are two phylogenetically closely related crop species, and this was not the case for the more distant P. vulgaris and G. max. Conversely, comparative mapping suggests that the convergent evolution of the indehiscent phenotype arose through mutations in different genes from the same underlying gene networks that are involved in secondary cell-wall biosynthesis and lignin deposition patterning at the pod level.

Decreased Nucleotide and Expression Diversity and Modified Coexpression Patterns Characterize Domestication in the Common Bean.

Using RNA sequencing technology and de novo transcriptome assembly, we compared representative sets of wild and domesticated accessions of common bean (Phaseolus vulgaris) from Mesoamerica. RNA was extracted at the first true-leaf stage, and de novo assembly was used to develop a reference transcriptome; the final data set consists of ∼190,000 single nucleotide polymorphisms from 27,243 contigs in expressed genomic regions. A drastic reduction in nucleotide diversity (∼60%) is evident for the domesticated form, compared with the wild form, and almost 50% of the contigs that are polymorphic were brought to fixation by domestication. In parallel, the effects of domestication decreased the diversity of gene expression (18%). While the coexpression networks for the wild and domesticated accessions demonstrate similar seminal network properties, they show distinct community structures that are enriched for different molecular functions. After simulating the demographic dynamics during domestication, we found that 9% of the genes were actively selected during domestication. We also show that selection induced a further reduction in the diversity of gene expression (26%) and was associated with 5-fold enrichment of differentially expressed genes. While there is substantial evidence of positive selection associated with domestication, in a few cases, this selection has increased the nucleotide diversity in the domesticated pool at target loci associated with abiotic stress responses, flowering time, and morphology.

Landscape genetics, adaptive diversity and population structure in Phaseolus vulgaris.

Here we studied the organization of genetic variation of the common bean (Phaseolus vulgaris) in its centres of domestication. We used 131 single nucleotide polymorphisms to investigate 417 wild common bean accessions and a representative sample of 160 domesticated genotypes, including Mesoamerican and Andean genotypes, for a total of 577 accessions. By analysing the genetic spatial patterns of the wild common bean, we documented the existence of several genetic groups and the occurrence of variable degrees of diversity in Mesoamerica and the Andes. Moreover, using a landscape genetics approach, we demonstrated that both demographic processes and selection for adaptation were responsible for the observed genetic structure. We showed that the study of correlations between markers and ecological variables at a continental scale can help in identifying local adaptation genes. We also located putative areas of common bean domestication in Mesoamerica, in the Oaxaca Valley, and the Andes, in southern Bolivia-northern Argentina. These observations are of paramount importance for the conservation and exploitation of the genetic diversity preserved within this species and other plant genetic resources.

Mesoamerican origin of the common bean (Phaseolus vulgaris L.) is revealed by sequence data.

Knowledge about the origins and evolution of crop species represents an important prerequisite for efficient conservation and use of existing plant materials. This study was designed to solve the ongoing debate on the origins of the common bean by investigating the nucleotide diversity at five gene loci of a large sample that represents the entire geographical distribution of the wild forms of this species. Our data clearly indicate a Mesoamerican origin of the common bean. They also strongly support the occurrence of a bottleneck during the formation of the Andean gene pool that predates the domestication, which was suggested by recent studies based on multilocus molecular markers. Furthermore, a remarkable result was the genetic structure that was seen for the Mesoamerican accessions, with the identification of four different genetic groups that have different relationships with the sets of wild accessions from the Andes and northern Peru-Ecuador. This finding implies that both of the gene pools from South America originated through different migration events from the Mesoamerican populations that were characteristic of central Mexico.

Molecular analysis of the parallel domestication of the common bean (Phaseolus vulgaris) in Mesoamerica and the Andes.

We have studied the nucleotide diversity of common bean, Phaseolus vulgaris, which is characterized by two independent domestications in two geographically distinct areas: Mesoamerica and the Andes. This provides an important model, as domestication can be studied as a replicate experiment. We used nucleotide data from five gene fragments characterized by large introns to analyse 214 accessions (102 wild and 112 domesticated). The wild accessions represent a cross-section of the entire geographical distribution of P. vulgaris. A reduction in genetic diversity in both of these gene pools was found, which was three-fold greater in Mesoamerica compared with the Andes. This appears to be a result of a bottleneck that occurred before domestication in the Andes, which strongly impoverished this wild germplasm, leading to the minor effect of the subsequent domestication bottleneck (i.e. sequential bottleneck). These findings show the importance of considering the evolutionary history of crop species as a major factor that influences their current level and structure of genetic diversity. Furthermore, these data highlight a single domestication event within each gene pool. Although the findings should be interpreted with caution, this evidence indicates the Oaxaca valley in Mesoamerica, and southern Bolivia and northern Argentina in South America, as the origins of common bean domestication.

Challenges and Opportunities behind the Use of Herbaria in Paleogenomics Studies.

Paleogenomics focuses on the recovery, manipulation, and analysis of ancient DNA (aDNA) from historical or long-dead organisms to reconstruct and analyze their genomes. The aDNA is commonly obtained from remains found in paleontological and archaeological sites, conserved in museums, and in other archival collections. Herbarium collections represent a great source of phenotypic and genotypic information, and their exploitation has allowed for inference and clarification of previously unsolved taxonomic and systematic relationships. Moreover, herbarium specimens offered a new source for studying phenological traits in plants and for disentangling biogeography and evolutionary scenarios of species. More recently, advances in molecular technologies went in parallel with the decreasing costs of next-generation sequencing (NGS) approaches, which paved the way to the utilization of aDNA for whole-genome studies. Although many studies have been carried out combining modern analytic techniques and ancient samples, such as herbarium specimens, this research field is still relatively unexplored due to the need for improving strategies for aDNA manipulation and exploitation from ancient samples. The higher susceptibility of aDNA to degradation and contamination during herbarium conservation and manipulation and the occurrence of biochemical postmortem damage can result in a more challenging reconstruction of the original DNA sequence. Here, we review the methodological approaches that have been developed for the exploitation of historical herbarium plant materials, such as best practices for aDNA extraction, amplification, and genotyping. We also focus on some strategies to overcome the main problems related to the utilization of herbarium specimens for their exploitation in plant evolutionary studies.

Selection and adaptive introgression guided the complex evolutionary history of the European common bean.

Domesticated crops have been disseminated by humans over vast geographic areas. Common bean (Phaseolus vulgaris L.) was introduced in Europe after 1492. Here, by combining whole-genome profiling, metabolic fingerprinting and phenotypic characterisation, we show that the first common bean cultigens successfully introduced into Europe were of Andean origin, after Francisco Pizarro's expedition to northern Peru in 1529. We reveal that hybridisation, selection and recombination have shaped the genomic diversity of the European common bean in parallel with political constraints. There is clear evidence of adaptive introgression into the Mesoamerican-derived European genotypes, with 44 Andean introgressed genomic segments shared by more than 90% of European accessions and distributed across all chromosomes except PvChr11. Genomic scans for signatures of selection highlight the role of genes relevant to flowering and environmental adaptation, suggesting that introgression has been crucial for the dissemination of this tropical crop to the temperate regions of Europe.

Açaí extract powder as natural antioxidant on pork patties during the refrigerated storage.

The current trends among consumers are pushing for the use of natural antioxidants options. Açaí fruit is rich on polyphenolic components but no studies have been carried out to evaluate their effect in meat products. The objective was to investigate the effect of açaí extract on refrigerated pork patties quality. Five treatments were done: without antioxidant (CON), Sodium Erythorbate 500 mg.kg -1 (ERY), Açaí Extract: 250 (AEL), 500 (AEM), 750 mg.kg -1 (AEH). Açaí extract did not affect the proximate composition, pH and cooking parameters. The concentrations of açaí extract studied increased antioxidant activity and reduced lipid oxidation (0.379, 0.293, and 0.217 vs. 0.889 mg MDA.kg-1 for AEL, AEM, AEH vs. CON, respectively). However, only the AEL treatment did not affect the color parameters, showing the best option for the application on pork patties. Thus, açaí extract at 250 mg.kg-1 can be used as a natural antioxidant replacing sodium erythorbate to preserve the quality of refrigerated pork patties.

Addition of Natural Extracts with Antioxidant Function to Preserve the Quality of Meat Products.

Antioxidants are used to prevent oxidation reactions and inhibit the development of unwanted sensory characteristics that decrease the nutritional quality, acceptance, and shelf-life of processed meat products, improving their stability. Synthetic antioxidants, although efficient, are related to the development of diseases because they present toxic and carcinogenic effects. Thus, researchers and the meat industry are studying natural alternatives to synthetic antioxidants to be used in meat products, thus meeting the demand of consumers who seek foods without additives in their composition. These natural extracts have compounds that exert antioxidant activity in different meat products by different mechanisms. Thus, this review work aimed to gather studies that applied natural extracts derived from different plant sources as possible antioxidants in meat products and their action in preserving the quality of these products.

Biodiversity studies in Phaseolus species by DNA barcoding.

The potential of DNA barcoding was tested as a system for studying genetic diversity and genetic traceability in bean germplasm. This technique was applied to several pure lines of Phaseolus vulgaris L. belonging to wild, domesticated, and cultivated common beans, along with some accessions of Phaseolus coccineus L., Phaseolus lunatus L., and Vigna unguiculata (L.) Walp. A multilocus approach was exploited using three chloroplast genic regions (rbcL, trnL, and matK), four intergenic spacers (rpoB-trnC, atpBrbcL, trnT-trnL, and psbA-trnH), and nuclear ITS1 and ITS2 rDNA sequences. Our main goals were to identify the markers and SNPs that show the best discriminant power at the variety level in common bean germplasm, to examine two methods (tree based versus character based) for biodiversity analysis and traceability assays, and to evaluate the overall utility of chloroplast DNA barcodes for reconstructing the origins of modern Italian varieties. Our results indicate that the neighbor-joining method is a powerful approach for comparing genetic diversity within plant species, but it is relatively uninformative for the genetic traceability of plant varieties. In contrast, the character-based method was able to identify several distinct haplotypes over all target regions corresponding to Mesoamerican or Andean accessions; Italian accessions originated from both gene pools. On the whole, our findings raise some concerns about the use of DNA barcoding for intraspecific genetic diversity studies in common beans and highlights its limitations for resolving genetic relationships between landraces and varieties.

Cytogenetic map of common bean (Phaseolus vulgaris L.).

A cytogenetic map of common bean was built by in situ hybridization of 35 bacterial artificial chromosomes (BACs) selected with markers mapping to eight linkage groups, plus two plasmids for 5S and 45S ribosomal DNA and one bacteriophage. Together with three previously mapped chromosomes (chromosomes 3, 4, and 7), 43 anchoring points between the genetic map and the cytogenetic map of the species are now available. Furthermore, a subset of four BAC clones was proposed to identify the 11 chromosome pairs of the standard cultivar BAT93. Three of these BACs labelled more than a single chromosome pair, indicating the presence of repetitive DNA in their inserts. A repetitive distribution pattern was observed for most of the BACs; for 38% of them, highly repetitive pericentromeric or subtelomeric signals were observed. These distribution patterns corresponded to pericentromeric and subtelomeric heterochromatin blocks observed with other staining methods. Altogether, the results indicate that around half of the common bean genome is heterochromatic and that genes and repetitive sequences are intermingled in the euchromatin and heterochromatin of the species.

Red pitaya extract as natural antioxidant in pork patties with total replacement of animal fat.

The antioxidant effects of red pitaya extract (PE) were evaluated in pork patties for 18 days at 2 °C. The following treatments were prepared: control (CON, without antioxidant), sodium erythorbate (ERY, 500 mg kg-1), PE low dose (PEL, 250 mg kg-1), PE medium dose (PEM, 500 mg kg-1), and PE high dose (PEH, 1000 mg kg-1). No significant effect was observed on chemical composition and cooking loss with the addition of PE, while a significant effect was noticed in cohesiveness (P < 0.05). The intense pink colour of PE enhanced the colour stability during storage (9.33, 7.92 and 7.69 vs. 6.77 for PEH, PEM and PEL vs. CON, respectively; (P < 0.05). TBARS (1.21 vs. 2.44 mg MDA/kg) and carbonyl values (5.45 vs. 6.87 nmol carbonyl/mg) of treated samples were lower than those observed in CON. Similar values were found between samples with PE and ERY. PE improved colour acceptance and the preference of pork patties. Therefore, PE is a very effective natural antioxidant by delaying colour and oxidative deterioration.

Strategies to increase the shelf life of meat and meat products with phenolic compounds.

Oxidative reactions and microbial growth are the main processes involved in the loss of quality in meat products. Although the use of additives to improve the shelf life is a common practice in the meat industry, the current trends among consumers are pushing the researchers and professionals of the meat industry to reformulate meat products. Polyphenols are compounds with antioxidant and antimicrobial activity naturally found in several plants, fruits, and vegetables that can be used in the production of extracts and components in active packaging to improve the shelf life of meat products. This chapter aims to discuss the advances in terms of (1) encapsulation techniques to protect phenolic compounds; (2) production of active and edible packages rich on phenolic compounds; (3) use of phenolic-rich additives (free or encapsulated form) with non-thermal technologies to improve the shelf life of meat products; and (4) use of active packaging rich on phenolic compounds on meat products. Innovative strategies to encapsulated polyphenols and produce films are mainly centered in the use of innovative and emerging technologies (such as ultrasound and supercritical fluids). Moreover, the combined use of polyphenols and non-thermal technologies is a relevant approach to improve the shelf life of meat products, especially using high pressure processing. In terms of application of innovative films, nanomaterials have been largely explored and indicated as relevant strategy to preserve meat and meat products.