Novel SNP markers for flowering and seed quality traits in faba bean (Vicia faba L.): characterization and GWAS of a diversity panel.

Faba bean (Vicia faba L.) is a legume crop grown in diverse climates worldwide. It has a high potential for increased cultivation to meet the need for more plant-based proteins in human diets, a prerequisite for a more sustainable food production system. Characterization of diversity panels of crops can identify variation in and genetic markers for target traits of interest for plant breeding. In this work, we collected a diversity panel of 220 accessions of faba bean from around the world consisting of gene bank material and commercially available cultivars. The aims of this study were to quantify the phenotypic diversity in target traits to analyze the impact of breeding on these traits, and to identify genetic markers associated with traits through a genome-wide association study (GWAS). Characterization under field conditions at Nordic latitude across two years revealed a large genotypic variation and high broad-sense heritability for eleven agronomic and seed quality traits. Pairwise correlations showed that seed yield was positively correlated to plant height, number of seeds per plant, and days to maturity. Further, susceptibility to bean weevil damage was significantly higher for early flowering accessions and accessions with larger seeds. In this study, no yield penalty was found for higher seed protein content, but protein content was negatively correlated to starch content. Our results showed that while breeding advances in faba bean germplasm have resulted in increased yields and number of seeds per plant, they have also led to a selection pressure towards delayed onset of flowering and maturity. DArTseq genotyping identified 6,606 single nucleotide polymorphisms (SNPs) by alignment to the faba bean reference genome. These SNPs were used in a GWAS, revealing 51 novel SNP markers significantly associated with ten of the assessed traits. Three markers for days to flowering were found in predicted genes encoding proteins for which homologs in other plant species regulate flowering. Altogether, this work enriches the growing pool of phenotypic and genotypic data on faba bean as a valuable resource for developing efficient breeding strategies to expand crop cultivation.

Estimates of heterozygosity from single nucleotide polymorphism markers are context-dependent and often wrong.

Genetic diversity is frequently described using heterozygosity, particularly in a conservation context. Often, it is estimated using single nucleotide polymorphisms (SNPs); however, it has been shown that heterozygosity values calculated from SNPs can be biased by both study design and filtering parameters. Though solutions have been proposed to address these issues, our own work has found them to be inadequate in some circumstances. Here, we aimed to improve the reliability and comparability of heterozygosity estimates, specifically by investigating how sample size and missing data thresholds influenced the calculation of autosomal heterozygosity (heterozygosity calculated from across the genome, i.e. fixed and variable sites). We also explored how the standard practice of tri- and tetra-allelic site exclusion could bias heterozygosity estimates and influence eventual conclusions relating to genetic diversity. Across three distinct taxa (a frog, Litoria rubella; a tree, Eucalyptus microcarpa; and a grasshopper, Keyacris scurra), we found heterozygosity estimates to be meaningfully affected by sample size and missing data thresholds, partly due to the exclusion of tri- and tetra-allelic sites. These biases were inconsistent both between species and populations, with more diverse populations tending to have their estimates more severely affected, thus having potential to dramatically alter interpretations of genetic diversity. We propose a modified framework for calculating heterozygosity that reduces bias and improves the utility of heterozygosity as a measure of genetic diversity, whilst also highlighting the need for existing population genetic pipelines to be adjusted such that tri- and tetra-allelic sites be included in calculations.

Intraspecific Variability of Wild-Growing Common Valerian (Valeriana officinalis L.).

Common valerian (Valeriana officinalis L.) is an important medicinal plant revealing sedative, hypotensive, anti-spasmodic and anxiolytic activity. The purpose of the study was to determine the intraspecific variability of the common valerian growing wild in Poland and the 'Lubelski' landrace, as to their developmental traits, chemical composition and selected genetic parameters. Both wild-growing populations (19) and the landrace (1) were evaluated under ex situ conditions. Observations of the underground organs parameters, both developmental and chemical (according to the European Pharmacopoeia) were carried out in the first year of the plant's development, while the characteristics of the aboveground organs, followed by the sowing value of seeds (according to the International Seed Testing Association)-in the second year. The genetic analyses were performed using the NGS-DArT-seq method. Results indicate the presence of five different gene pools covering the regions of population's origin, with a gene flow within and between them. A high level of developmental and chemical variabilities among the wild-growing populations was noticed, however without a clear relation to the region of the origin. The mass of underground organs ranged from 107.4 to 403.6 g FW × plant-1 with the content of sesquiterpenic acids at the level of 0.004-0.094%. Population no 18 was distinguished by the highest content of sesquiterpenic acids and the relatively high mass of underground organs, followed by the admixture of the gene pool, typical for the 'Lubelski' landrace. Unlike the 'Lubelski' landrace, the wild-growing populations were characterized by a high amount of an essential oils (3.90 to 10.04 mL/kg), which may be promising from the perspective of their potential use. In turn, the sowing value of the seeds obtained from the populations, expressed as the germinability, was rather low (25.25-62.25%).

Nitrogen Fertilizer Application Alters the Root Endophyte Bacterial Microbiome in Maize Plants, but Not in the Stem or Rhizosphere Soil.

Plant-associated microorganisms that affect plant development, their composition, and their functionality are determined by the host, soil conditions, and agricultural practices. How agricultural practices affect the rhizosphere microbiome has been well studied, but less is known about how they might affect plant endophytes. In this study, the metagenomic DNA from the rhizosphere and endophyte communities of root and stem of maize plants was extracted and sequenced with the "diversity arrays technology sequencing," while the bacterial community and functionality (organized by subsystems from general to specific functions) were investigated in crops cultivated with or without tillage and with or without N fertilizer application. Tillage had a small significant effect on the bacterial community in the rhizosphere, but N fertilizer had a highly significant effect on the roots, but not on the rhizosphere or stem. The relative abundance of many bacterial species was significantly different in the roots and stem of fertilized maize plants, but not in the unfertilized ones. The abundance of N cycle genes was affected by N fertilization application, most accentuated in the roots. How these changes in bacterial composition and N genes composition might affect plant development or crop yields has still to be unraveled. IMPORTANCE We investigated the bacterial community structure in the rhizosphere, root, and stem of maize plants cultivated under different agricultural techniques, i.e., with or without N fertilization, and with or without tillage. We found that the bacterial community was defined mostly by the plant compartment and less by agricultural techniques. In the roots, N fertilizer application affected the bacterial community structure, the microbiome functionality, and the abundance of genes involved in the N cycle, but the effect in the rhizosphere and stem was much smaller. Contrary, tillage did not affect the maize microbiome. This study enriches our knowledge about the plant-microbiome system and how N fertilization application affected it.

High gene flow in the silverlip pearl oyster Pinctada maxima between inshore and offshore sites near Eighty Mile Beach in Western Australia.

An understanding of stock recruitment dynamics in fisheries is fundamental to successful management. Pinctada maxima is a bivalve mollusc widely distributed throughout the Indo-Pacific and is the main species targeted for cultured pearl and pearl shell production in Australia. Pearl production in Australia relies heavily on wild-caught individuals, the majority of which come from the Eighty Mile Beach region near Broome in Western Australia. In this study, we used a genotyping by sequencing approach to explore fine-scale patterns of genetic connectivity among inshore shallow and offshore deep populations of P. maxima near Eighty Mile Beach. Our results revealed high-levels of gene flow among inshore and offshore sites and no differences in genetic diversity between depths. Global estimates of genetic differentiation were low (F ST = 0.006) but significantly different from zero, and pairwise estimates of genetic differentiation among sites were significant in only 3% of comparisons. Moreover, Bayesian clustering detected no separation of inshore and offshore sample sites, and instead showed all samples to be admixed among sites, locations and depths. Despite an absence of any clear spatial clustering among sites, we identified a significant pattern of isolation by distance. In a dynamic environment like Eighty Mile Beach, genetic structure can change from year-to-year and successive dispersal and recruitment events over generations likely act to homogenize the population. Although we cannot rule out the null hypothesis of panmixia, our data indicate high levels of dispersal and connectivity among inshore and offshore fishing grounds.

Improved Methods for Deamination-Based m6A Detection.

N 6-methyladenosine (m6A) is a critical regulator of gene expression and cellular function. Much of our knowledge of m6A has been enabled by the identification of m6A sites transcriptome-wide. However, global m6A profiling methods require high amounts of input RNA to accurately identify methylated RNAs, making m6A profiling from rare cell types or scarce tissue samples infeasible. To overcome this issue, we previously developed DART-seq, which relies on the expression of a fusion protein consisting of the APOBEC1 cytidine deaminase tethered to the m6A-binding YTH domain. APOBEC1-YTH directs C-to-U mutations adjacent to m6A sites, therefore enabling single nucleotide-resolution m6A mapping. Here, we present an improved version of DART-seq which utilizes a variant of the YTH domain engineered to achieve enhanced m6A recognition. In addition, we develop in vitro DART-seq and show that it performs similarly to cellular DART-seq and can map m6A in any sample of interest using nanogram amounts of total RNA. Altogether, these improvements to the DART-seq approach will enable better m6A detection and will facilitate the mapping of m6A in samples not previously amenable to global m6A profiling.

Detecting m6A with In Vitro DART-Seq.

Recent studies have uncovered that cellular mRNAs contain a diverse epitranscriptome comprising chemically modified bases which play important roles in gene expression regulation. Among these is m6A, which is a highly prevalent modification that contributes to several aspects of RNA regulation and cellular function. Traditional methods for m6A profiling have used m6A antibodies to immunoprecipitate methylated RNAs. Although powerful, such methods require high amounts of input material. Recently, we developed DART-seq, an antibody-free method for m6A profiling from low-input RNA samples. DART-seq relies on deamination of cytidines that invariably follow m6A sites and can be performed using a simple in vitro assay with only 50 ng of total RNA. Here, we describe the in vitro DART method and present a detailed protocol for highly sensitive m6A profiling from any RNA sample of interest.

Diversity of Water Yam (Dioscorea alata L.) Accessions from Côte d'Ivoire Based on SNP Markers and Agronomic Traits.

Dioscorea alata (L.), also referred to as water, winged, or greater yam, is one of the most economically important staple food crops in tropical and subtropical areas. In Côte d'Ivoire, it represents, along with other yam species, the largest food crop and significantly contributes to food security. However, studies focusing on better understanding the structure and extent of genetic diversity among D. alata accessions, using molecular and phenotypic traits, are limited. This study was, therefore, conducted to assess the pattern of genetic variability in a set of 188 D. alata accessions from the National Agronomic Research Centre (CNRA) genebank using 11,722 SNP markers (generated by the Diversity Arrays Technology) and nine agronomic traits. Phylogenetic analyses using hierarchical clustering, admixture, kinship, and Discriminant analysis of principal component (DAPC) all assigned the accessions into four main clusters. Genetic diversity assessment using molecular-based SNP markers showed a high proportion of polymorphic SNPs (87.81%). The analysis of molecular variance (AMOVA) showed low molecular variability within genetic groups. In addition, the agronomic traits evaluated for two years in field conditions showed a high heritability and high variability among D. alata accessions. This study provides insights into the genetic diversity among accessions in the CNRA genebank and opens an avenue for sustainable resource management and the identification of promising parental clones for water yam breeding programs in Côte d'Ivoire.

Genotypic Variation in Cultivated and Wild Sorghum Genotypes in Response to Striga hermonthica Infestation.

Striga hermonthica is the most important parasitic weed in sub-Saharan Africa and remains one of the most devastating biotic factors affecting sorghum production in the western regions of Kenya. Farmers have traditionally managed Striga using cultural methods, but the most effective and practical solution to poor smallholder farmers is to develop Striga-resistant varieties. This study was undertaken with the aim of identifying new sources of resistance to Striga in comparison with the conventional sources as standard checks. We evaluated 64 sorghum genotypes consisting of wild relatives, landraces, improved varieties, and fourth filial generation (F4) progenies in both a field trial and a pot trial. Data were collected for days to 50% flowering (DTF), dry panicle weight (DPW, g), plant height (PH, cm), yield (YLD, t ha-1), 100-grain weight (HGW, g), overall disease score (ODS), overall pest score (OPS), area under Striga number progress curve (ASNPC), maximum above-ground Striga (NSmax), and number of Striga-forming capsules (NSFC) at relevant stages. Genetic diversity and hybridity confirmation was determined using Diversity Arrays Technology sequencing (DArT-seq). Residual heterosis for HGW and NSmax was calculated as the percent increase or decrease in performance of F4 crossover midparent (MP). The top 10 best yielding genotypes were predominantly F4 crosses in both experiments, all of which yielded better than resistant checks, except FRAMIDA in the field trial and HAKIKA in the pot trial. Five F4 progenies (ICSVIII IN × E36-1, LANDIWHITE × B35, B35 × E36-1, F6YQ212 × B35, and ICSVIII IN × LODOKA) recorded some of the highest HGW in both trials revealing their stability in good performance. Three genotypes (F6YQ212, GBK045827, and F6YQ212xB35) and one check (SRN39) were among the most resistant to Striga in both trials. SNPs generated from DArT-seq grouped the genotypes into three major clusters, with all resistant checks grouping in the same cluster except N13. We identified more resistant and high-yielding genotypes than the conventional checks, especially among the F4 crosses, which should be promoted for adoption by farmers. Future studies will need to look for more diverse sources of Striga resistance and pyramid different mechanisms of resistance into farmer-preferred varieties to enhance the durability of Striga resistance in the fields of farmers.

Assessment of the Genetic Structure and Diversity of Soybean (Glycine max L.) Germplasm Using Diversity Array Technology and Single Nucleotide Polymorphism Markers.

Knowledge of the genetic structure and diversity of germplasm collections is crucial for sustainable genetic improvement through hybridization programs and rapid adaptation to changing breeding objectives. The objective of this study was to determine the genetic diversity and population structure of 281 International Institute of Tropical Agriculture (IITA) soybean accessions using diversity array technology (DArT) and single nucleotide polymorphism (SNP) markers for the efficient utilization of these accessions. From the results, the SNP and DArT markers were well distributed across the 20 soybean chromosomes. The cluster and principal component analyses revealed the genetic diversity among the 281 accessions by grouping them into two stratifications, a grouping that was also evident from the population structure analysis, which divided the 281 accessions into two distinct groups. The analysis of molecular variance revealed that 97% and 98% of the genetic variances using SNP and DArT markers, respectively, were within the population. Genetic diversity indices such as Shannon's diversity index, diversity and unbiased diversity revealed the diversity among the different populations of the soybean accessions. The SNP and DArT markers used provided similar information on the structure, diversity and polymorphism of the accessions, which indicates the applicability of the DArT marker in genetic diversity studies. Our study provides information about the genetic structure and diversity of the IITA soybean accessions that will allow for the efficient utilization of these accessions in soybean improvement programs, especially in Africa.

High-Density Mapping of Triple Rust Resistance in Barley Using DArT-Seq Markers.

The recent availability of an assembled and annotated genome reference sequence for the diploid crop barley (Hordeum vulgare L.) provides new opportunities to study the genetic basis of agronomically important traits such as resistance to stripe [Puccinia striiformis f. sp. hordei (Psh)], leaf [P. hordei (Ph)], and stem [P. graminis f. sp. tritici (Pgt)] rust diseases. The European barley cultivar Pompadour is known to possess high levels of resistance to leaf rust, predominantly due to adult plant resistance (APR) gene Rph20. We developed a barley recombinant inbred line (RIL) population from a cross between Pompadour and the leaf rust and stripe rust susceptible selection Biosaline-19 (B-19), and genotyped this population using DArT-Seq genotyping by sequencing (GBS) markers. In the current study, we produced a high-density linkage map comprising 8,610 (SNP and in silico) markers spanning 5957.6 cM, with the aim of mapping loci for resistance to leaf rust, stem rust, and stripe rust. The RIL population was phenotyped in the field with Psh (Mexico and Ecuador) and Ph (Australia) and in the greenhouse at the seedling stage with Australian Ph and Pgt races, and at Wageningen University with a European variant of Psh race 24 (PshWUR). For Psh, we identified a consistent field QTL on chromosome 2H across all South American field sites and years. Two complementary resistance genes were mapped to chromosomes 1H and 4H at the seedling stage in response to PshWUR, likely to be the loci rpsEm1 and rpsEm2 previously reported from the cultivar Emir from which Pompadour was bred. For leaf rust, we determined that Rph20 in addition to two minor-effect QTL on 1H and 3H were effective at the seedling stage, whilst seedling resistance to stem rust was due to QTL on chromosomes 3H and 7H conferred by Pompadour and B-19, respectively.

Characterizing Palestinian snake melon (Cucumis melo var. flexuosus) germplasm diversity and structure using SNP and DArTseq markers.

BACKGROUND: Crop landraces embody a source of beneficial genes potentially providing endurance to environmental stress and other agronomic qualities including yield. Our study included 88 snake melon accessions (Cucumis melo var. flexuosus) collected from 9 districts in the Palestinian West-Bank. These accessions represent four landraces of Palestinian snake melon: Green, and White Baladi, and Green, and White Sahouri. RESULTS: This is the first report on successful application of genotyping by sequencing in snake melon. Nine thousand seven hundred fifty single-nucleotide polymorphism (SNP) and 7400 DArTseq genetic markers were employed to evaluate genetic biodiversity and population structure of Palestinian snake melon germplasm collection. Clustering based on neighbor-joining-analysis, principle coordinate and Bayesian model implemented in Structure showed that patterns of genetic diversity of snake melon landraces depends on their geographical source and unraveled the presence of two major local landraces (Sahouri, and Baladi) with accessions from each group clustering together. A significant correlation was observed between both types of markers in Mantel correlation test. A significant association between genetic and geographic matrices (P < 0.0001) was also detected. AMOVA indicated that majority of variation (90%) was due to the difference within accessions. CONCLUSION: The Palestinian landraces seem to have unique genes that may allow the enhancement of the global snake melon gene pool and developments of the plant production worldwide. Our subsequent objective is to detect genotypes with promising qualities and to conduct association mapping studies concentrating on Fusarium-wilt resistance, yield, and environmental stresses.

Efficient anchoring of alien chromosome segments introgressed into bread wheat by new Leymus racemosus genome-based markers.

BACKGROUND: The tertiary gene pool of bread wheat, to which Leymus racemosus belongs, has remained underutilized due to the current limited genomic resources of the species that constitute it. Continuous enrichment of public databases with useful information regarding these species is, therefore, needed to provide insights on their genome structures and aid successful utilization of their genes to develop improved wheat cultivars for effective management of environmental stresses. RESULTS: We generated de novo DNA and mRNA sequence information of L. racemosus and developed 110 polymorphic PCR-based markers from the data, and to complement the PCR markers, DArT-seq genotyping was applied to develop additional 9990 SNP markers. Approximately 52% of all the markers enabled us to clearly genotype 22 wheat-L. racemosus chromosome introgression lines, and L. racemosus chromosome-specific markers were highly efficient in detailed characterization of the translocation and recombination lines analyzed. A further analysis revealed remarkable transferability of the PCR markers to three other important Triticeae perennial species: L. mollis, Psathyrostachys huashanica and Elymus ciliaris, indicating their suitability for characterizing wheat-alien chromosome introgressions carrying chromosomes of these genomes. CONCLUSION: The efficiency of the markers in characterizing wheat-L. racemosus chromosome introgression lines proves their reliability, and their high transferability further broadens their scope of application. This is the first report on sequencing and development of markers from L. racemosus genome and the application of DArT-seq to develop markers from a perennial wild relative of wheat, marking a paradigm shift from the seeming concentration of the technology on cultivated species. Integration of these markers with appropriate cytogenetic methods would accelerate development and characterization of wheat-alien chromosome introgression lines.

Population structure of Chinese southwest wheat germplasms resistant to stripe rust and powdery mildew using the DArT-seq technique / População de germoplasmas resistentes de trigo empregando a técnica de sequenciamento Dart-seq

ABSTRACT: Understanding genetic variability in existing wheat accessions is critical for collection, conservation and use of wheat germplasms. In this study, 138 Chinese southwest wheat accessions were investigated by genotyping using two resistance gene makers (Pm21 and Yr26) and DArT-seq technique. Finally, about 50% cultivars (lines) amplified the specific allele for the Yr26 gene (Gwm11) and 40.6% for the Pm21 gene (SCAR1265). By DArT-seq analysis, 30,485 markers (6486 SNPs and 23999 DArTs) were obtained with mean polymorphic information content (PIC) value 0.33 and 0.28 for DArT and SNP marker, respectively. The mean Dice genetic similarity coefficient (GS) was 0.72. Two consistent groups of wheat varieties were identified using principal coordinate analysis (PCoA) at the level of both the chromosome 6AS and the whole-genome, respectively. Group I was composed of non-6VS/6AL translocation lines of different origins, while Group II was composed of 6VS/6AL translocation (T6VS/6AL) lines, most of which carried the Yr26 and Pm21 genes and originated from Guizhou. Besides, a model-based population structure analysis revealed extensive admixture and further divided these wheat accessions into six subgroups (SG1, SG2, SG3, SG4, SG5 and SG6), based on their origin, pedigree or disease resistance. This information is useful for wheat breeding in southwestern China and association mapping for disease resistance using these wheat germplasms in future.

RESUMO: O conhecimento da estrutura da população é essencial para o mapeamento de associação de resistência a doenças para a população de trigo. Neste estudo, a técnica de DART-seq™ foi usada para genotipar o genoma inteiro de cultivares de trigo. Finalmente, 30,485 marcadores (6486 SNPs e 23999 dardos) foram obtidos, e dois grupos de variedades de trigo foram identificados por meio de análise principal-coordenadas (PCoA) do nível de todo o genoma e o nível 6AS cromossomo. O grupo I foi composto por linhas não T6VS/6Al de diferentes origens, enquanto o Grupo II foi composto de linhas T6VS/6Al, sendo que da maioria destes realizados os genes Yr26 e PM21 originários de Guizhou.

A genome-wide association study of 23 agronomic traits in Chinese wheat landraces.

Uncovering the genetic basis of agronomic traits in wheat landraces is important for ensuring global food security via the development of improved varieties. Here, 723 wheat landraces from 10 Chinese agro-ecological zones were evaluated for 23 agronomic traits in six environments. All accessions could be clustered into five subgroups based on phenotypic data via discriminant function analysis, which was highly consistent with genotypic classification. A genome-wide association study was conducted for these traits using 52 303 DArT-seq markers to identify marker-trait associations and candidate genes. Using both the general linear model and the mixed linear model, 149 significant markers were identified for 21 agronomic traits based on best linear unbiased prediction values. Considering the linkage disequilibrium decay distance in this study, significant markers within 10 cM were combined as a quantitative trait locus (QTL), with a total of 29 QTL identified for 15 traits. Of these, five QTL for heading date, flag leaf width, peduncle length, and thousand kernel weight had been reported previously. Twenty-five candidate genes associated with significant markers were identified. These included the known vernalization genes VRN-B1 and vrn-B3 and the photoperiod response genes Ppd and PRR. Overall, this study should be helpful in elucidating the underlying genetic mechanisms of complex agronomic traits and performing marker-assisted selection in wheat.