Large and non-spherical seeds are less likely to form a persistent soil seed bank.

There is some evidence that seed traits can affect the long-term persistence of seeds in the soil. However, findings on this topic have differed between systems. Here, we brought together a worldwide database of seed persistence data for 1474 species to test the generality of seed mass-shape-persistence relationships. We found a significant trend for low seed persistence to be associated with larger and less spherical seeds. However, the relationship varied across different clades, growth forms and species ecological preferences. Specifically, relationships of seed mass-shape-persistence were more pronounced in Poales than in other order clades. Herbaceous species that tend to be found in sites with low soil sand content and precipitation have stronger relationships between seed shape and persistence than in sites with higher soil sand content and precipitation. For the woody plants, the relationship between persistence and seed morphology was stronger in sites with high soil sand content and low precipitation than in sites with low soil sand content and higher precipitation. Improving the ability to predict the soil seed bank formation process, including burial and persistence, could benefit the utilization of seed morphology-persistence relationships in management strategies for vegetation restoration and controlling species invasion across diverse vegetation types and environments.

Phenotypic characterization and candidate gene analysis of a short kernel and brassinosteroid insensitive mutant from hexaploid oat (Avena sativa).

In an ethyl methanesulfonate oat (Avena sativa) mutant population we have found a mutant with striking differences to the wild-type (WT) cv. Belinda. We phenotyped the mutant and compared it to the WT. The mutant was crossed to the WT and mapping-by-sequencing was performed on a pool of F2 individuals sharing the mutant phenotype, and variants were called. The impacts of the variants on genes present in the reference genome annotation were estimated. The mutant allele frequency distribution was combined with expression data to identify which among the affected genes was likely to cause the observed phenotype. A brassinosteroid sensitivity assay was performed to validate one of the identified candidates. A literature search was performed to identify homologs of genes known to be involved in seed shape from other species. The mutant had short kernels, compact spikelets, altered plant architecture, and was found to be insensitive to brassinosteroids when compared to the WT. The segregation of WT and mutant phenotypes in the F2 population was indicative of a recessive mutation of a single locus. The causal mutation was found to be one of 123 single-nucleotide polymorphisms (SNPs) spanning the entire chromosome 3A, with further filtering narrowing this down to six candidate genes. In-depth analysis of these candidate genes and the brassinosteroid sensitivity assay suggest that a Pro303Leu substitution in AVESA.00010b.r2.3AG0419820.1 could be the causal mutation of the short kernel mutant phenotype. We identified 298 oat proteins belonging to orthogroups of previously published seed shape genes, with AVESA.00010b.r2.3AG0419820.1 being the only of these affected by a SNP in the mutant. The AVESA.00010b.r2.3AG0419820.1 candidate is functionally annotated as a GSK3/SHAGGY-like kinase with homologs in Arabidopsis, wheat, barley, rice, and maize, with several of these proteins having known mutants giving rise to brassinosteroid insensitivity and shorter seeds. The substitution in AVESA.00010b.r2.3AG0419820.1 affects a residue with a known gain-of function substitution in Arabidopsis BRASSINOSTEROID-INSENSITIVE2. We propose a gain-of-function mutation in AVESA.00010b.r2.3AG0419820.1 as the most likely cause of the observed phenotype, and name the gene AsGSK2.1. The findings presented here provide potential targets for oat breeders, and a step on the way towards understanding brassinosteroid signaling, seed shape and nutrition in oats.

Seed shape and size of Silene latifolia, differences between sexes, and influence of the parental genome in hybrids with Silene dioica.

Introduction: Plants undergo various natural changes that dramatically modify their genomes. One is polyploidization and the second is hybridization. Both are regarded as key factors in plant evolution and result in phenotypic differences in different plant organs. In Silene, we can find both examples in nature, and this genus has a seed shape diversity that has long been recognized as a valuable source of information for infrageneric classification. Methods: Morphometric analysis is a statistical study of shape and size and their covariations with other variables. Traditionally, seed shape description was limited to an approximate comparison with geometric figures (rounded, globular, reniform, or heart-shaped). Seed shape quantification has been based on direct measurements, such as area, perimeter, length, and width, narrowing statistical analysis. We used seed images and processed them to obtain silhouettes. We performed geometric morphometric analyses, such as similarity to geometric models and elliptic Fourier analysis, to study the hybrid offspring of S. latifolia and S. dioica. Results: We generated synthetic tetraploids of Silene latifolia and performed controlled crosses between diploid S. latifolia and Silene dioica to analyze seed morphology. After imaging capture and post-processing, statistical analysis revealed differences in seed size, but not in shape, between S. latifolia diploids and tetraploids, as well as some differences in shape among the parentals and hybrids. A detailed inspection using fluorescence microscopy allowed for the identification of shape differences in the cells of the seed coat. In the case of hybrids, differences were found in circularity and solidity. Overal seed shape is maternally regulated for both species, whereas cell shape cannot be associated with any of the sexes. Discussion: Our results provide additional tools useful for the combination of morphology with genetics, ecology or taxonomy. Seed shape is a robust indicator that can be used as a complementary tool for the genetic and phylogenetic analyses of Silene hybrid populations.

Dormancy, germination, and associated seed ecological traits of 25 Fabaceae species from northern India.

Fabaceae produce seeds with water-impermeable seed coats, i.e., physical dormancy (PY). We hypothesized that the proportion of PY seeds will increase with the dryness of the habitat, and some key seed ecological traits will be strongly associated with different levels of PY. Fresh seed of 25 Fabaceae species collected in northern India were used for imbibition and germination experiments to determine the proportion of seeds with PY and of nondormant (ND) seeds compared to their Sri Lankan congeners. Seed coat:seed mass ratio (SCR), 1000 seed weight, seed shape index (SSI), embryo type and median germination time of ND seeds were determined. Four imbibition and germination patterns were identified among seeds of the studied species. Seeds collected from Indian populations had a higher proportion of PY seeds than those of Sri Lankan populations. We identified a type of embryo called 'spatulate axile' that had not been identified before among the studied species. Species with ND seeds had a lower SCR and a higher SSI than those with PY. Our hypothesis was confirmed since populations from drier habitats in India produce a higher proportion of PY seeds than those from Sri Lanka. A low SCR ensures minimal resistance to germinating seeds, while seeds with a high SSI have a lower tendency to incorporate into the soil seed bank. Thus, these seed traits aid the fast germination of ND seeds, often dispersed just before the rainy season.

Characterization of Invasiveness, Thermotolerance and Light Requirement of Nine Invasive Species in China.

Understanding responsible functional traits for promoting plant invasiveness could be important to aid in the development of adequate management strategies for invasive species. Seed traits play an important role in the plant life cycle by affecting dispersal ability, formation of the soil seed bank, type and level of dormancy, germination, survival and/or competitive ability. We assessed seed traits and germination strategies of nine invasive species under five temperature regimes and light/dark treatments. Our results showed a considerable level of interspecific variation in germination percentage among the tested species. Both cooler (5/10 °C) and warmer (35/40 °C) temperatures tended to inhibit germination. All study species were considered small-seeded, and seed size did not affect germination in the light. Yet, a slightly negative correlation was found between germination in the dark and seed dimensions. We classified the species into three categories according to their germination strategies: (i) risk-avoiders, mostly displaying dormant seeds with low G%; (ii) risk-takers, reaching a high G% in a broad range of temperatures; (iii) intermediate species, showing moderate G% values, which could be enhanced in specific temperature regimes. Variability in germination requirements could be important to explain species coexistence and invasion ability of plants to colonize different ecosystems.

Genetic mapping and genome-wide association study identify BhYAB4 as the candidate gene regulating seed shape in wax gourd (Benincasa hispida).

Wax gourd is an important vegetable crop of the Cucurbitaceae family. According to the shape and structure of the seed coat, the seeds of the wax gourd can be divided into bilateral and unilateral. Bilateral seeds usually germinate quickly and have a high germination rate than unilateral seeds. Thereby, wax gourd varieties with bilateral seeds are more welcomed by seed companies and growers. However, the genetic basis and molecular mechanism regulating seed shape remain unclear in the wax gourd. In this study, the genetic analysis demonstrated that the seed shape of wax gourd was controlled by a single gene, with bilateral dominant to unilateral. Combined with genetic mapping and genome-wide association study, Bhi04G000544 (BhYAB4), encoding a YABBY transcription factor, was identified as the candidate gene for seed shape determination in the wax gourd. A G/A single nucleotide polymorphism variation of BhYAB4 was detected among different germplasm resources, with BhYAB4G specifically enriched in bilateral seeds and BhYAB4A in unilateral seeds. The G to A mutation caused intron retention and premature stop codon of BhYAB4. Expression analysis showed that both BhYAB4G and BhYAB4A were highly expressed in seeds, while the nuclear localization of BhYAB4A protein was disturbed compared with that of BhYAB4G protein. Finally, a derived cleaved amplified polymorphic sequence marker that could efficiently distinguish between bilateral and unilateral seeds was developed, thereby facilitating the molecular marker-assisted breeding of wax gourd cultivars.

QTL Analysis for Bread Wheat Seed Size, Shape and Color Characteristics Estimated by Digital Image Processing.

The size, shape, and color of wheat seeds are important traits that are associated with yield and flour quality (size, shape), nutritional value, and pre-harvest sprouting (coat color). These traits are under multigenic control, and to dissect their molecular and genetic basis, quantitative trait loci (QTL) analysis is used. We evaluated 114 recombinant inbred lines (RILs) in a bi-parental RIL mapping population (the International Triticeae Mapping Initiative, ITMI/MP) grown in 2014 season. We used digital image analysis for seed phenotyping and obtained data for seven traits describing seed size and shape and 48 traits of seed coat color. We identified 212 additive and 34 pairs of epistatic QTLs on all the chromosomes of wheat genome except chromosomes 1A and 5D. Many QTLs were overlapping. We demonstrated that the overlap between QTL regions was low for seed size/shape traits and high for coat color traits. Using the literature and KEGG data, we identified sets of genes in Arabidopsis and rice from the networks controlling seed size and color. Further, we identified 29 and 14 candidate genes for seed size-related loci and for loci associated with seed coat color, respectively.

New Geometric Models for Shape Quantification of the Dorsal View in Seeds of Silene Species.

The description of shape in Silene seeds is based on adjectives coined by naturalists in the 19th century. The expressions reniform, dorso plana, and dorso canaliculata were applied in reference to lateral or dorsal views of seeds, but the characters described can be submitted now to an analytical description by quantitative methods, allowing shape quantification and the comparison between species or populations. A quantitative morphological analysis is based on the comparison with geometric models that adjust to the shape of seeds. Morphological analysis of the dorsal view of Silene seeds based on geometric models is applied here to 26 seed populations belonging to 12 species. According to their dorsal views, the seeds are classified as convex and non-convex. New geometric models are presented for both types, including figures such as super-ellipses and modified ellipses. The values of J index (percent of similarity of a seed image with the model) are obtained in representative seed samples from diverse populations and species. The quantitative description of seed shape based on the comparison with geometric models allows the study of variation in shape between species and in populations, as well as the identification of seeds in Silene species. The method is of application to other plant species.

Vertical distribution of soil seed bank and the ecological importance of deeply buried seeds in alkaline grasslands.

Background: Soil seed banks play a central role in vegetation dynamics and may be an important source of ecological restoration. However, the vast majority of seed bank studies examined only the uppermost soil layers (0-10 cm); hence, our knowledge on the depth distribution of seed bank and the ecological significance of deeply buried seeds is limited. The aim of our study was to examine the fine-scale vertical distribution of soil seed bank to a depth of 80 cm, which is one of the largest studied depth gradients so far. Our model systems were alkaline grasslands in East-Hungary, characterised by harsh environmental conditions, due to Solonetz soil reference group with Vertic horizon. We asked the following questions: (1) How do the seedling density and species richness of soil seed bank change along a vertical gradient and to what depth can germinable seeds be detected? (2) What is the relationship between the depth distribution of the germinable seeds and the species traits? Methods: In each of the five study sites, four soil cores (4 cm diameter) of 80 cm depth were collected with an auger for soil seed bank analysis. Each sample was divided into sixteen 5-cm segments by depth (320 segments in total). Samples were concentrated by washing over sieves and then germinated in an unheated greenhouse. Soil penetration resistance was measured in situ next to each core location (0-80 cm depth, 1-cm resolution). We tested the number and species richness of seedlings observed in the soil segments (N = 320), using negative binomial generalized linear regression models, in which sampling layer and penetration resistance were the predictor variables. We ran the models for morphological groups (graminoids/forbs), ecological groups (grassland species/weeds) and life-form categories (short-lived/perennial). We also tested whether seed shape index, seed mass, water requirement or salt tolerance of the species influence the vertical distribution of their seed bank. Results: Germinable seed density and species richness in the seed bank decreased with increasing soil depth and penetration resistance. However, we detected nine germinable seeds of six species even in the deepest soil layer. Forbs, grassland species and short-lived species occurred in large abundance in deep layers, from where graminoids, weeds and perennial species were missing. Round-shaped seeds were more abundant in deeper soil layers compared to elongated ones, but seed mass and ecological indicator values did not influence the vertical seed bank distribution. Our research draws attention to the potential ecological importance of the deeply buried seeds that may be a source of recovery after severe disturbance. As Vertisols cover 335 million hectares worldwide, these findings can be relevant for many regions and ecosystems globally. We highlight the need for similar studies in other soil and habitat types to test whether the presence of deep buried seeds is specific to soils with Vertic characteristics.

Identification of quantitative trait loci (QTLs) and candidate genes for seed shape and 100-seed weight in soybean [Glycine max (L.) Merr.].

Seed size and shape are important traits determining yield and quality in soybean. Seed size and shape are also desirable for specialty soy foods like tofu, natto, miso, and edamame. In order to find stable quantitative trait loci (QTLs) and candidate genes for seed shape and 100-seed weight, the current study used vegetable type and seed soybean-derived F2 and F2:3 mapping populations. A total of 42 QTLs were mapped, which were dispersed across 13 chromosomes. Of these, seven were determined to be stable QTLs and five of them were major QTLs, namely qSL-10-1, qSW-4-1, qSV-4-1, qSLW-10-1, and qSLH-10-1. Thirteen of the 42 QTLs detected in the current study were found at known loci, while the remaining 29 were discovered for the first time. Out of these 29 novel QTLs, 17 were major QTLs. Based on Protein Analysis Through Evolutionary Relationships (PANTHER), gene annotation information, and literature search, 66 genes within seven stable QTLs were predicted to be possible candidate genes that might regulate seed shape and seed weight in soybean. The current study identified the key candidate genes and quantitative trait loci (QTLs) controlling soybean seed shape and weight, and these results will be very helpful in marker-assisted breeding for developing soybean varieties with improved seed weight and desired seed shape.

Cannabis Varieties Can Be Distinguished by Achene Shape Using Geometric Morphometrics.

Introduction: Cannabis plant uses are widespread across human cultures. The current tendency is to classify Cannabis varieties into chemovars upon their chemical fingerprint, mainly cannabinoids and terpenoids content. The identification of chemovars has important medical implications; however, their pharmacological characterization is costly and time consuming. The goal of this study was to assess whether achene shape variation could be related to Cannabis varieties with contrasting cannabinoid concentrations, as a first approach to chemovar identification. Methods: We used two-dimensional geometric morphometrics (GM) of the achenes and multivariate statistical analysis. We used achenes from five varieties, two from Type II chemotype (expressing both tetrahydrocannabinol [THC] and cannabidiol [CBD]), two Type I (THC-only), and one Type III (CBD-only). Results: The achenes from the different chemotypes were clearly distinguishable. No significant differences between varieties from the same chemotype were observed. The varieties with high THC concentration (Type I) were rounded and bigger, whereas achene from varieties containing only CBD (Type III) had a slender shape with smaller size. Conclusion: Achene shape variation is a potential biomarker of cannabinoid content in the plant flowers. Further studies are needed to confirm the suitability of GM methods for high-throughput screening of Cannabis cultivars, including larger diversity of varieties, and taking into account growth conditions, which can also influence plant chemical fingerprint.

Geometric Models for Seed Shape Description and Quantification in the Cactaceae.

Seed shape in species of the Cactaceae is described by comparison with geometric models. Three new groups of models are presented, two for symmetric seeds, and a third group for asymmetric seeds. The first two groups correspond, respectively, to superellipses and the combined equations of two semi-ellipses. The third group contains models derived from the representation of polar equations of Archimedean spirals that define the shape of asymmetric seeds in genera of different subfamilies. Some of the new models are geometric curves, while others are composed with a part resulting from the average silhouettes of seeds. The application of models to seed shape quantification permits the analysis of variation in seed populations, as well as the comparison of shape between species. The embryos of the Cactaceae are of the peripheral type, strongly curved and in contact with the inner surface of the seed coat. A relationship is found between seed elongation and the models, in which the genera with elongated seeds are represented by models with longer trajectories of the spiral. The analysis of seed shape opens new opportunities for taxonomy and allows quantification of seed shape in species of the Cactaceae.

Seed Geometry in the Vitaceae.

The Vitaceae Juss., in the basal lineages of Rosids, contains sixteen genera and 950 species, mainly of tropical lianas. The family has been divided in five tribes: Ampelopsideae, Cisseae, Cayratieae, Parthenocisseae and Viteae. Seed shape is variable in this family. Based on new models derived from equations representing heart and water drop curves, we describe seed shape in species of the Vitaceae. According to their similarity to geometric models, the seeds of the Vitaceae have been classified in ten groups. Three of them correspond to models before described and shared with the Arecaceae (lenses, superellipses and elongated water drops), while in the seven groups remaining, four correspond to general models (waterdrops, heart curves, elongated heart curves and other elongated models) and three adjust to the silhouettes of seeds in particular genera (heart curves of Cayratia and Pseudocayratia, heart curves of the Squared Heart Curve (SqHC) type of Ampelocissus and Ampelopsis and Elongated Superellipse-Heart Curves (ESHCs), frequent in Tetrastigma species and observed also in Cissus species and Rhoicissus rhomboidea). The utilities of the application of geometric models for seed description and shape quantification in this family are discussed.

A spinach genome assembly with remarkable completeness, and its use for rapid identification of candidate genes for agronomic traits.

Spinach (Spinacia oleracea) is grown as a nutritious leafy vegetable worldwide. To accelerate spinach breeding efficiency, a high-quality reference genome sequence with great completeness and continuity is needed as a basic infrastructure. Here, we used long-read and linked-read technologies to construct a de novo spinach genome assembly, designated SOL_r1.1, which was comprised of 287 scaffolds (total size: 935.7 Mb; N50 = 11.3 Mb) with a low proportion of undetermined nucleotides (Ns = 0.34%) and with high gene completeness (BUSCO complete 96.9%). A genome-wide survey of resistance gene analogues identified 695 genes encoding nucleotide-binding site domains, receptor-like protein kinases, receptor-like proteins and transmembrane-coiled coil domains. Based on a high-density double-digest restriction-site associated DNA sequencing-based linkage map, the genome assembly was anchored to six pseudomolecules representing ∼73.5% of the whole genome assembly. In addition, we used SOL_r1.1 to identify quantitative trait loci for bolting timing and fruit/seed shape, which harbour biologically plausible candidate genes, such as homologues of the FLOWERING LOCUS T and EPIDERMAL PATTERNING FACTOR-LIKE genes. The new genome assembly, SOL_r1.1, will serve as a useful resource for identifying loci associated with important agronomic traits and for developing molecular markers for spinach breeding/selection programs.

A Major and Stable Quantitative Trait Locus qSS2 for Seed Size and Shape Traits in a Soybean RIL Population.

Seed size and shape traits are important determinants of seed yield and appearance quality in soybean [Glycine max (L.) Merr.]. Understanding the genetic architecture of these traits is important to enable their genetic improvement through efficient and targeted selection in soybean breeding, and for the identification of underlying causal genes. To map seed size and shape traits in soybean, a recombinant inbred line (RIL) population developed from K099 (small seed size) × Fendou 16 (large seed size), was phenotyped in three growing seasons. A genetic map of the RIL population was developed using 1,485 genotyping by random amplicon sequencing-direct (GRAS-Di) and 177 SSR markers. Quantitative trait locus (QTL) mapping was conducted by inclusive composite interval mapping. As a result, 53 significant QTLs for seed size traits and 27 significant QTLs for seed shape traits were identified. Six of these QTLs (qSW8.1, qSW16.1, qSLW2.1, qSLT2.1, qSWT1.2, and qSWT4.3) were identified with LOD scores of 3.80-14.0 and R 2 of 2.36%-39.49% in at least two growing seasons. Among the above significant QTLs, 24 QTLs were grouped into 11 QTL clusters, such as, three major QTLs (qSL2.3, qSLW2.1, and qSLT2.1) were clustered into a major QTL on Chr.02, named as qSS2. The effect of qSS2 was validated in a pair of near isogenic lines, and its candidate genes (Glyma.02G269400, Glyma.02G272100, Glyma.02G274900, Glyma.02G277200, and Glyma.02G277600) were mined. The results of this study will assist in the breeding programs aiming at improvement of seed size and shape traits in soybean.

The APETALA2 transcription factor LsAP2 regulates seed shape in lettuce.

Seeds are major vehicles of propagation and dispersal in plants. A number of transcription factors, including APETALA2 (AP2), play crucial roles during the seed development process in various plant species. However, genes essential for seed development and the regulatory networks that operate during seed development remain unclear in lettuce. Here, we identified a lettuce AP2 (LsAP2) gene that was highly expressed during the early stages of seed development. LsAP2 knockout plants obtained by the CRISPR/Cas9 system were used to explore the biological function of LsAP2. Compared with the wild type, the seeds of Lsap2 mutant plants were longer and narrower, and developed an extended tip at the seed top. After further investigating the structural characteristics of the seeds of Lsap2 mutant plants, we proposed a new function of LsAP2 in seed dispersal. Moreover, we identified several interactors of LsAP2. Our results showed that LsAP2 directly interacted with the lettuce homolog of BREVIPEDICELLUS (LsBP) and promoted the expression of LsBP. Transcriptome analysis revealed that LsAP2 might also be involved in brassinosteroid biosynthesis and signaling pathways. Taken together, our data indicate that LsAP2 has a significant function in regulating seed shape in lettuce.

Seed Morphology in Silene Based on Geometric Models.

Seed description in morphology is often based on adjectives such as "spherical", "globular", or "reniform", but this does not provide a quantitative method. A new morphological approach based on the comparison of seed images with geometric models provides a seed description in Silene species on a quantitative basis. The novelty of the proposed method is based in the comparison of the seed images with geometric models according to a cardioid shape. The J index is a measurement that indicates the seed percentage of similarity with a cardioid or cardioid-derived figures used as models. The seeds of Silene species have high values of similarity with the cardioid and cardioid-derived models (J index superior to 90). The comparison with different figures allows species description and differentiation. The method is applied here to seeds of 21 species and models are proposed for some of them including S. diclinis, an endangered species. The method is discussed in the context of previous comparison with the measures used in traditional morphometric analysis. The similarity of seed images with geometric figures opens a new perspective for the automatized taxonomical evaluation of samples linking seed morphology to functional traits in endangered Silene species.

Identification of Additive-Epistatic QTLs Conferring Seed Traits in Soybean Using Recombinant Inbred Lines.

Seed weight and shape are important agronomic traits that affect soybean quality and yield. In the present study, we used image analysis software to evaluate 100-seed weight and seed shape traits (length, width, perimeter, projection area, length/width, and weight/projection area) of 155 novel recombinant inbred soybean lines (NJRISX) generated by crossing "Su88-M21" and "XYXHD". We examined quantitative trait loci (QTLs) associated with the six traits (except seed weight per projection area), and identified 42 additive QTLs (5-8 QTLs per trait) accounting for 24.9-37.5% of the phenotypic variation (PV). Meanwhile, 2-4 epistatic QTL pairs per trait out of a total of 18 accounted for 2.5-7.2% of the PV; and unmapped minor QTLs accounted for the remaining 35.0-56.7% of the PV. A total of 28 additive and 11 epistatic QTL pairs were concentrated in nine joint QTL segments (JQSs), indicating that QTLs associated with seed weight and shape are closely related and interacted. An interaction was also detected between additive and epistatic QTL pairs and environment, which made significant contributions of 1.4-9.5% and 0.4-0.8% to the PV, respectively. We annotated 18 candidate genes in the nine JQSs, which were important for interpreting the close relationships among the six traits. These findings indicate that examining the interactions between closely related traits rather than only analyzing individual trait provides more useful insight into the genetic system of the interrelated traits for which there has been limited QTL information.

High-Resolution Mapping in Two RIL Populations Refines Major "QTL Hotspot" Regions for Seed Size and Shape in Soybean (Glycine max L.).

Seed size and shape are important traits determining yield and quality in soybean. However, the genetic mechanism and genes underlying these traits remain largely unexplored. In this regard, this study used two related recombinant inbred line (RIL) populations (ZY and K3N) evaluated in multiple environments to identify main and epistatic-effect quantitative trait loci (QTLs) for six seed size and shape traits in soybean. A total of 88 and 48 QTLs were detected through composite interval mapping (CIM) and mixed-model-based composite interval mapping (MCIM), respectively, and 15 QTLs were common among both methods; two of them were major (R2 > 10%) and novel QTLs (viz., qSW-1-1ZN and qSLT-20-1K3N). Additionally, 51 and 27 QTLs were identified for the first time through CIM and MCIM methods, respectively. Colocalization of QTLs occurred in four major QTL hotspots/clusters, viz., "QTL Hotspot A", "QTL Hotspot B", "QTL Hotspot C", and "QTL Hotspot D" located on Chr06, Chr10, Chr13, and Chr20, respectively. Based on gene annotation, gene ontology (GO) enrichment, and RNA-Seq analysis, 23 genes within four "QTL Hotspots" were predicted as possible candidates, regulating soybean seed size and shape. Network analyses demonstrated that 15 QTLs showed significant additive x environment (AE) effects, and 16 pairs of QTLs showing epistatic effects were also detected. However, except three epistatic QTLs, viz., qSL-13-3ZY, qSL-13-4ZY, and qSW-13-4ZY, all the remaining QTLs depicted no main effects. Hence, the present study is a detailed and comprehensive investigation uncovering the genetic basis of seed size and shape in soybeans. The use of a high-density map identified new genomic regions providing valuable information and could be the primary target for further fine mapping, candidate gene identification, and marker-assisted breeding (MAB).

MuSeeQ, a novel supervised image analysis tool for the simultaneous phenotyping of the soluble mucilage and seed morphometric parameters.

BACKGROUND: The mucilage is a model to study the polysaccharide biosynthesis since it is produced in large amounts and composed of complex polymers. In addition, it is of great economic interest for its technical and nutritional value. A fast method for phenotyping the released mucilage and the seed morphometric parameters will be useful for fundamental, food, pharmaceutical and breeding researches. Current strategies to phenotype soluble mucilage are restricted to visual evaluations or are highly time-consuming. RESULTS: Here, we developed a high-throughput phenotyping method for the simultaneous measurement of the soluble mucilage content released on a gel and the seed morphometric parameters. Within this context, we combined a biochemical assay and an open-source computer-aided image analysis tool, MuSeeQ. The biochemical assay consists in sowing seeds on an agarose medium containing the dye toluidine blue O, which specifically stains the mucilage once it is released on the gel. The second part of MuSeeQ is a macro developed in ImageJ allowing to quickly extract and analyse 11 morphometric data of seeds and their respective released mucilages. As an example, MuSeeQ was applied on a flax recombinant inbred lines population (previously screened for fatty acids content.) and revealed significant correlations between the soluble mucilage shape and the concentration of some fatty acids, e.g. C16:0 and C18:2. Other fatty acids were also found to correlate with the seed shape parameters, e.g. C18:0 and C18:2. MuSeeQ was then showed to be used for the analysis of other myxospermous species, including Arabidopsis thaliana and Camelina sativa. CONCLUSIONS: MuSeeQ is a low-cost and user-friendly method which may be used by breeders and researchers for phenotyping simultaneously seeds of specific cultivars, natural variants or mutants and their respective soluble mucilage area released on a gel. The script of MuSeeQ and video tutorials are freely available at http://MuSeeQ.free.fr.