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.

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.

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.

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.

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.

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.

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.

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.

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.

Characterization of Nutritional Quality Traits of a Common Bean Germplasm Collection.

Food legumes are at the crossroads of many societal challenges that involve agriculture, such as climate change and food sustainability and security. In this context, pulses have a crucial role in the development of plant-based diets, as they represent a very good source of nutritional components and improve soil fertility, such as by nitrogen fixation through symbiosis with rhizobia. The main contribution to promotion of food legumes in agroecosystems will come from plant breeding, which is guaranteed by the availability of well-characterized genetic resources. Here, we analyze seeds of 25 American and European common bean purified accessions (i.e., lines of single seed descent) for different morphological and compositional quality traits. Significant differences among the accessions and superior genotypes for important nutritional traits are identified, with some lines showing extreme values for more than one trait. Heritability estimates indicate the importance of considering the effects of environmental growth conditions on seed compositional traits. They suggest the need for more phenotypic characterization in different environments over different years to better characterize combined effects of environment and genotype on nutritional trait variations. Finally, adaptation following the introduction and spread of common bean in Europe seems to have affected its nutritional profile. This finding further suggests the relevance of evolutionary studies to guide breeders in the choice of plant genetic resources.

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.

Towards Development, Maintenance, and Standardized Phenotypic Characterization of Single-Seed-Descent Genetic Resources for Lupins.

Well-characterized genetic resources are fundamental to maintain and provide the various genotypes for pre-breeding programs for the production of new cultivars (e.g., wild relatives, unimproved material, landraces). The aim of the current article is to provide protocols for the characterization of the genetic resources of two lupin crop species: the European Lupinus albus and the American Lupinus mutabilis. Intelligent nested collections of lupins derived from homozygous lines (single-seed descent) are being developed, established, and exploited using cutting-edge approaches for genotyping, phenotyping, data management, and data analysis within the INCREASE project (EU Horizon 2020). This will allow us to predict the phenotypic performance of genotyped lines, and will further boost research and development in lupins. Lupins stand out due to their high-quality seed protein (∼40% of seed dry weight) and other primary components in the seeds, which include fatty acids, dietary fiber, and minerals. The potential of lupins as a crop is highlighted by the multiple benefits of plant-based food in terms of food security, nutrition, human health, and sustainable production. The use of lupins in foods, along with other well-studied and widely used food legumes, will also provide a greatly diversified plant-based food palette to meet the Global Goals for Sustainable Development to improve people's lives by 2030. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Lupin seed phenotypic descriptors Basic Protocol 2: Lupin seed imaging Basic Protocol 3: Standardized phenotypic characterization of lupin genetic resources grown towards primary seed increase (development of single-seed descent genetic resources).

Towards the Development, Maintenance, and Standardized Phenotypic Characterization of Single-Seed-Descent Genetic Resources for Common Bean.

The optimal use of legume genetic resources represents a key prerequisite for coping with current agriculture-related societal challenges, including conservation of agrobiodiversity, agricultural sustainability, food security, and human health. Among legumes, the common bean (Phaseolus vulgaris) is the most economically important for human consumption, and its evolutionary trajectories as a species have been crucial to determining the structure and level of its present and available genetic diversity. Genomic advances are considerably enhancing the characterization and assessment of important genetic variants. For this purpose, the development and availability of, and access to, well-described and efficiently managed genetic resource collections that comprise pure lines derived by single-seed-descent cycles will be paramount for the use of the reservoir of common bean variability and for the advanced breeding of legume crops. This is one of the main aims of the new and challenging European project INCREASE, which is the implementation of Intelligent Collections with appropriate standardized protocols that must be characterized, maintained, and made available, along with the related data, to users such as breeders and researchers. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Characterizing common bean seeds for seed trait descriptors Basic Protocol 2: Bean seed imaging Basic Protocol 3: Characterizing bean lines for plant trait descriptors specific for common bean Primary Seed Increase.

Ancient genomes reveal early Andean farmers selected common beans while preserving diversity.

All crops are the product of a domestication process that started less than 12,000 years ago from one or more wild populations1,2. Farmers selected desirable phenotypic traits (such as improved energy accumulation, palatability of seeds and reduced natural shattering3) while leading domesticated populations through several more or less gradual demographic contractions2,4. As a consequence, the erosion of wild genetic variation5 is typical of modern cultivars, making them highly susceptible to pathogens, pests and environmental change6,7. The loss of genetic diversity hampers further crop improvement programmes to increase food production in a changing world, posing serious threats to food security8,9. Using both ancient and modern seeds, we analysed the temporal dynamics of genetic variation and selection during the domestication process of the common bean (Phaseolus vulgaris) in the southern Andes. Here, we show that most domestic traits were selected for before 2,500 years ago, with no or only minor loss of whole-genome heterozygosity. In fact, most of the changes at coding genes and linked regions that differentiate wild and domestic genomes are already present in the ancient genomes analysed here, and all ancient domestic genomes dated between 600 and 2,500 years ago are highly variable (at least as variable as modern genomes from the wild). Single seeds from modern cultivars show reduced variation when compared with ancient seeds, indicating that intensive selection within cultivars in the past few centuries probably partitioned ancestral variation within different genetically homogenous cultivars. When cultivars from different Andean regions are pooled, the genomic variation of the pool is higher than that observed in the pool of ancient seeds from north and central western Argentina. Considering that most desirable phenotypic traits are probably controlled by multiple polymorphic genes10, a plausible explanation of this decoupling of selection and genetic erosion is that early farmers applied a relatively weak selection pressure2 by using many phenotypically similar but genetically diverse individuals as parents. Our results imply that selection strategies during the past few centuries, as compared with earlier times, more intensively reduced genetic variation within cultivars and produced further improvements by focusing on a few plants carrying the traits of interest, at the cost of marked genetic erosion within Andean landraces.

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.

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.

Use of Turkey Meat Affected by White Striping Myopathy for the Development of Low-Fat Cooked Sausage Enriched with Chitosan.

The main objective of this research was the development of a healthy meat product from turkey meat with white striping myopathy. The effect of adding different proportions of chitosan on the qualitative characteristics, sensory acceptance, and stability of cooked sausages during storage was studied. Three treatments were elaborated (control, 1.5% chitosan, and 3% chitosan), stored for 56 days, and characterized in terms of chemical composition, texture profile analysis, drip and pressure loss analysis, and sensory analysis (after processing; day 0). In the different storage periods (0 and 56 days), the pH value, color, thiobarbituric acid reactive substances (TBARS), and volatile compounds were evaluated. The results showed that the moisture content, lipids, proteins, and weight loss decreased (p < 0.05) and the ash content increased (p < 0.05) with the addition of chitosan. Similarly, the values of texture parameters (hardness, cohesiveness, gumminess, and chewiness) were higher in the sausages reformulated with chitosan than in control samples. The addition of chitosan increased the pH and yellowness (b*) values and reduced (p < 0.05) redness (a*) and lightness (L*) values. The b* values (only in reformulated sausages) and pH increased during storage, while a* showed a significant reduction after 56 storage days. Lipid oxidation (TBARS) was kept below the limits of quantification in all samples and both after processing and 56 storage days. However, when quantifying the lipid-derived volatiles, a clear antioxidant activity of chitosan was observed, which limits the release of these compounds, mainly aldehydes (hexanal and nonanal). Finally, the sensory analysis indicated that, although chitosan treatments received the lowest scores for all attributes, the reformulated samples did not differ from control sausages. Therefore, sausage containing chitosan may represent an interesting alternative for adding value to turkey meats affected by white striping myopathy and, at the same time, develop into a healthy and functional meat product increasing the proportion of fibers in one's diet.

GWAS Based on RNA-Seq SNPs and High-Throughput Phenotyping Combined with Climatic Data Highlights the Reservoir of Valuable Genetic Diversity in Regional Tomato Landraces.

Tomato (Solanum lycopersicum L.) is a widely used model plant species for dissecting out the genomic bases of complex traits to thus provide an optimal platform for modern "-omics" studies and genome-guided breeding. Genome-wide association studies (GWAS) have become a preferred approach for screening large diverse populations and many traits. Here, we present GWAS analysis of a collection of 115 landraces and 11 vintage and modern cultivars. A total of 26 conventional descriptors, 40 traits obtained by digital phenotyping, the fruit content of six carotenoids recorded at the early ripening (breaker) and red-ripe stages and 21 climate-related variables were analyzed in the context of genetic diversity monitored in the 126 accessions. The data obtained from thorough phenotyping and the SNP diversity revealed by sequencing of ripe fruit transcripts of 120 of the tomato accessions were jointly analyzed to determine which genomic regions are implicated in the expressed phenotypic variation. This study reveals that the use of fruit RNA-Seq SNP diversity is effective not only for identification of genomic regions that underlie variation in fruit traits, but also of variation related to additional plant traits and adaptive responses to climate variation. These results allowed validation of our approach because different marker-trait associations mapped on chromosomal regions where other candidate genes for the same traits were previously reported. In addition, previously uncharacterized chromosomal regions were targeted as potentially involved in the expression of variable phenotypes, thus demonstrating that our tomato collection is a precious reservoir of diversity and an excellent tool for gene discovery.

Impact of ultrasound and potassium chloride on the physicochemical and sensory properties in low sodium restructured cooked ham.

The objectives of this study were to evaluate the effects of power ultrasound (nominal intensity 600 W·cm-2 for 10 min) and the addition of potassium chloride (KCl) on the physicochemical properties and sensorial acceptance of low sodium restructured cooked ham. Four treatments of low sodium restructured cooked ham (mean of 324.52 mg Na/100 g) were prepared: CT - Control Treatment; UsT - Ultrasound Treatment; KT - addition of 0.5% KCl; UsKT - Ultrasound Treatment and addition of 0.5% KCl. Ultrasound application reduced the total fluid released and improved the sensory acceptance for salty taste and flavor compared to CT. The addition of KCl showed the lowest values for total fluid release, the highest scores for all parameters of sensory acceptance, improved hardness and chewiness, which results were not statistically different from the results obtained by combining ultrasound and KCl. Therefore, the use of KCl was considered a technological and sensorial viable alternative to produce low sodium restructured cooked ham. CHEMICAL COMPOUNDS USED IN THIS RESEARCH: Methanol (PubChem CID: 887); Chloroform (PubChem CID: 6212); Sodium Carbonate (PubChem CID: 10340); Sodium hydroxide (PubChem CID: 14798); Boric acid (PubChem CID: 7628).