Expanded functionality, increased accuracy, and enhanced speed in the de novo genotyping-by-sequencing pipeline GBS-SNP-CROP.

SUMMARY: GBS-SNP-CROP is a bioinformatics pipeline originally developed to support the cost-effective genome-wide characterization of plant genetic resources through paired-end genotyping-by-sequencing (GBS), particularly in the absence of a reference genome. Since its 2016 release, the pipeline's functionality has greatly expanded, its computational efficiency has improved, and its applicability to a broad set of genomic studies for both plants and animals has been demonstrated. This note details the suite of improvements to date, as realized in GBS-SNP-CROP v.4.0, with specific attention paid to a new integrated metric that facilitates reliable variant identification despite the complications of homologs. Using the new de novo GBS read simulator GBS-Pacecar, also introduced in this note, results show an improvement in overall pipeline accuracy from 66% (v.1.0) to 84% (v.4.0), with a time saving of ∼70%. Both GBS-SNP-CROP versions significantly outperform TASSEL-UNEAK; and v.4.0 resolves the issue of non-overlapping variant calls observed between UNEAK and v.1.0. AVAILABILITY AND IMPLEMENTATION: GBS-SNP-CROP source code and user manual are available at https://github.com/halelab/GBS-SNP-CROP. The GBS read simulator GBS-Pacecar is available at https://github.com/halelab/GBS-Pacecar. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

'apparent': a simple and flexible R package for accurate SNP-based parentage analysis in the absence of guiding information.

BACKGROUND: The accurate determination of parent-progeny relationships within both in situ natural populations and ex situ genetic resource collections can greatly enhance plant breeding/domestication efforts and support plant genetic resource conservation strategies. Although a range of parentage analysis tools are available, none are designed to infer such relationships using genome-wide single nucleotide polymorphism (SNP) data in the complete absence of guiding information, such as generational groups, partial pedigrees, or genders. The R package ('apparent') developed and presented here addresses this gap. RESULTS: 'apparent' adopts a novel strategy of parentage analysis based on a test of genetic identity between a theoretically expected progeny (EPij), whose genotypic state can be inferred at all homozygous loci for a pair of putative parents (i and j), and all potential offspring (POk), represented by the k individuals of a given germplasm collection. Using the Gower Dissimilarity metric (GD), genetic identity between EPij and POk is taken as evidence that individuals i and j are the true parents of offspring k. Significance of a given triad (parental pairij + offspringk) is evaluated relative to the distribution of all GDij|k values for the population. With no guiding information provided, 'apparent' correctly identified the parental pairs of 15 lines of known pedigree within a test population of 77 accessions of Actinidia arguta, a performance unmatched by five other commonly used parentage analysis tools. In the case of an inconclusive triad analysis due to the absence of one parent from the test population, 'apparent' can perform a subsequent dyad analysis to identify a likely single parent for a given offspring. Average dyad analysis accuracy was 73.3% in the complete absence of pedigree information but increased to 100% when minimal generational information (adults vs. progeny) was provided. CONCLUSIONS: The 'apparent' R package is a fast and accurate parentage analysis tool that uses genome-wide SNP data to identify parent-progeny relationships within populations for which no a priori knowledge of family structure exists.

Mapping non-host resistance to the stem rust pathogen in an interspecific barberry hybrid.

BACKGROUND: Non-host resistance (NHR) presents a compelling long-term plant protection strategy for global food security, yet the genetic basis of NHR remains poorly understood. For many diseases, including stem rust of wheat [causal organism Puccinia graminis (Pg)], NHR is largely unexplored due to the inherent challenge of developing a genetically tractable system within which the resistance segregates. The present study turns to the pathogen's alternate host, barberry (Berberis spp.), to overcome this challenge. RESULTS: In this study, an interspecific mapping population derived from a cross between Pg-resistant Berberis thunbergii (Bt) and Pg-susceptible B. vulgaris was developed to investigate the Pg-NHR exhibited by Bt. To facilitate QTL analysis and subsequent trait dissection, the first genetic linkage maps for the two parental species were constructed and a chromosome-scale reference genome for Bt was assembled (PacBio + Hi-C). QTL analysis resulted in the identification of a single 13 cM region (~ 5.1 Mbp spanning 13 physical contigs) on the short arm of Bt chromosome 3. Differential gene expression analysis, combined with sequence variation analysis between the two parental species, led to the prioritization of several candidate genes within the QTL region, some of which belong to gene families previously implicated in disease resistance. CONCLUSIONS: Foundational genetic and genomic resources developed for Berberis spp. enabled the identification and annotation of a QTL associated with Pg-NHR. Although subsequent validation and fine mapping studies are needed, this study demonstrates the feasibility of and lays the groundwork for dissecting Pg-NHR in the alternate host of one of agriculture's most devastating pathogens.

The role of genetics in mainstreaming the production of new and orphan crops to diversify food systems and support human nutrition.

Especially in low-income nations, new and orphan crops provide important opportunities to improve diet quality and the sustainability of food production, being rich in nutrients, capable of fitting into multiple niches in production systems, and relatively adapted to low-input conditions. The evolving space for these crops in production systems presents particular genetic improvement requirements that extensive gene pools are able to accommodate. Particular needs for genetic development identified in part with plant breeders relate to three areas of fundamental importance for addressing food production and human demographic trends and associated challenges, namely: facilitating integration into production systems; improving the processability of crop products; and reducing farm labour requirements. Here, we relate diverse involved target genes and crop development techniques. These techniques include transgressive methods that involve defining exemplar crop models for effective new and orphan crop improvement pathways. Research on new and orphan crops not only supports the genetic improvement of these crops, but they serve as important models for understanding crop evolutionary processes more broadly, guiding further major crop evolution. The bridging position of orphan crops between new and major crops provides unique opportunities for investigating genetic approaches for de novo domestications and major crop 'rewildings'.

An interspecific barberry hybrid enables genetic dissection of non-host resistance to the stem rust pathogen Puccinia graminis.

Stem rust, caused by Puccinia graminis (Pg), remains a devastating disease of wheat, and the emergence of new Pg races virulent on deployed resistance genes fuels the ongoing search for sources of durable resistance. Despite its intrinsic durability, non-host resistance (NHR) is largely unexplored as a protection strategy against Pg, partly due to the inherent challenge of developing a genetically tractable system within which NHR segregates. Here, we demonstrate that Pg's far less studied ancestral host, barberry (Berberis spp.), provides such a unique pathosystem. Characterization of a natural population of B. ×ottawensis, an interspecific hybrid of Pg-susceptible B. vulgaris and Pg-resistant B. thunbergii (Bt), reveals that this uncommon nothospecies can be used to dissect the genetic mechanism(s) of Pg-NHR exhibited by Bt. Artificial inoculation of a natural population of B. ×ottawensis accessions, verified via genotyping by sequencing to be first-generation hybrids, revealed 51% susceptible, 33% resistant, and 16% intermediate phenotypes. Characterization of a B. ×ottawensis full sib family excluded the possibility of maternal inheritance of the resistance. By demonstrating segregation of Pg-NHR in a hybrid population, this study challenges the assumed irrelevance of Bt to Pg epidemiology and lays a novel foundation for the genetic dissection of NHR to one of agriculture's most studied pathogens.

Identification and characterization of Rht25, a locus on chromosome arm 6AS affecting wheat plant height, heading time, and spike development.

KEY MESSAGE: This study identified Rht25, a new plant height locus on wheat chromosome arm 6AS, and characterized its pleiotropic effects on important agronomic traits. Understanding genes regulating wheat plant height is important to optimize harvest index and maximize grain yield. In modern wheat varieties grown under high-input conditions, the gibberellin-insensitive semi-dwarfing alleles Rht-B1b and Rht-D1b have been used extensively to confer lodging tolerance and improve harvest index. However, negative pleiotropic effects of these alleles (e.g., poor seedling emergence and reduced biomass) can cause yield losses in hot and dry environments. As part of current efforts to diversify the dwarfing alleles used in wheat breeding, we identified a quantitative trait locus (QHt.ucw-6AS) affecting plant height in the proximal region of chromosome arm 6AS (< 0.4 cM from the centromere). Using a large segregating population (~ 2800 gametes) and extensive progeny tests (70-93 plants per recombinant family), we mapped QHt.ucw-6AS as a Mendelian locus to a 0.2 cM interval (144.0-148.3 Mb, IWGSC Ref Seq v1.0) and show that it is different from Rht18. QHt.ucw-6AS is officially designated as Rht25, with Rht25a representing the height-increasing allele and Rht25b the dwarfing allele. The average dwarfing effect of Rht25b was found to be approximately half of the effect observed for Rht-B1b and Rht-D1b, and the effect is greater in the presence of the height-increasing Rht-B1a and Rht-D1a alleles than in the presence of the dwarfing alleles. Rht25b is gibberellin-sensitive and shows significant pleiotropic effects on coleoptile length, heading date, spike length, spikelet number, spikelet density, and grain weight. Rht25 represents a new alternative dwarfing locus that should be evaluated for its potential to improve wheat yield in different environments.

GBS-SNP-CROP: a reference-optional pipeline for SNP discovery and plant germplasm characterization using variable length, paired-end genotyping-by-sequencing data.

BACKGROUND: With its simple library preparation and robust approach to genome reduction, genotyping-by-sequencing (GBS) is a flexible and cost-effective strategy for SNP discovery and genotyping, provided an appropriate reference genome is available. For resource-limited curation, research, and breeding programs of underutilized plant genetic resources, however, even low-depth references may not be within reach, despite declining sequencing costs. Such programs would find value in an open-source bioinformatics pipeline that can maximize GBS data usage and perform high-density SNP genotyping in the absence of a reference. RESULTS: The GBS SNP-Calling Reference Optional Pipeline (GBS-SNP-CROP) developed and presented here adopts a clustering strategy to build a population-tailored "Mock Reference" from the same GBS data used for downstream SNP calling and genotyping. Designed for libraries of paired-end (PE) reads, GBS-SNP-CROP maximizes data usage by eliminating unnecessary data culling due to imposed read-length uniformity requirements. Using 150 bp PE reads from a GBS library of 48 accessions of tetraploid kiwiberry (Actinidia arguta), GBS-SNP-CROP yielded on average three times as many SNPs as TASSEL-GBS analyses (32 and 64 bp tag lengths) and over 18 times as many as TASSEL-UNEAK, with fewer genotyping errors in all cases, as evidenced by comparing the genotypic characterizations of biological replicates. Using the published reference genome of a related diploid species (A. chinensis), the reference-based version of GBS-SNP-CROP behaved similarly to TASSEL-GBS in terms of the number of SNPs called but had an improved read depth distribution and fewer genotyping errors. Our results also indicate that the sets of SNPs detected by the different pipelines above are largely orthogonal to one another; thus GBS-SNP-CROP may be used to augment the results of alternative analyses, whether or not a reference is available. CONCLUSIONS: By achieving high-density SNP genotyping in populations for which no reference genome is available, GBS-SNP-CROP is worth consideration by curators, researchers, and breeders of under-researched plant genetic resources. In cases where a reference is available, especially if from a related species or when the target population is particularly diverse, GBS-SNP-CROP may complement other reference-based pipelines by extracting more information per sequencing dollar spent. The current version of GBS-SNP-CROP is available at https://github.com/halelab/GBS-SNP-CROP.git.

QTLs for uniform grain dimensions and germination selected during wheat domestication are co-located on chromosome 4B.

KEY MESSAGE: A major locus on the long arm of wheat chromosome 4B controls within-spikelet variation in both grain size and seed dormancy, the latter an important survival mechanism likely eliminated from wild wheat during domestication. Seed dormancy can increase the probability of survival of at least some progeny under unstable environmental conditions. In wild emmer wheat, only one of the two grains in a spikelet germinates during the first rainy season following maturation; and this within-plant variation in seed dormancy is associated with both grain dimension differences and position within the spikelet. Here, in addition to characterizing these associations, we elucidate the genetic mechanism controlling differential grain dimensions and dormancy within wild tetraploid wheat spikelets using phenotypic data from a wild emmer × durum wheat population and a high-density genetic map. We show that in wild emmer, the lower grain within the spikelet is about 30 % smaller and more dormant than the larger, upper grain that germinates usually within 3 days. We identify a major locus on the long arm of chromosome 4B that explains >40 % of the observed variation in grain dimensions and seed dormancy within spikelets. This locus, designated QGD-4BL, is validated using an independent set of wild emmer × durum wheat genetic stocks. The domesticated variant of this novel locus on chromosome 4B, likely fixed during the process of wheat domestication, favors spikelets with seeds of uniform size and synchronous germination. The identification of locus QGD-4BL enhances our knowledge of the genetic basis of the domestication syndrome of one of our most important crops.

Mapping a region within the 1RS.1BL translocation in common wheat affecting grain yield and canopy water status.

KEY MESSAGE: This study identifies a small distal region of the 1RS chromosome from rye that has a positive impact on wheat yield. The translocation of the short arm of rye (Secale cereale L.) chromosome one (1RS) onto wheat (Triticum aestivum L.) chromosome 1B (1RS.1BL) is used in wheat breeding programs worldwide due to its positive effect on yield, particularly under abiotic stress. Unfortunately, this translocation is associated with poor bread-making quality. To mitigate this problem, the 1RS arm was engineered by the removal and replacement of two interstitial rye segments with wheat chromatin: a distal segment to introduce the Glu-B3/Gli-B1 loci from wheat, and a proximal segment to remove the rye Sec-1 locus. We used this engineered 1RS chromosome (henceforth 1RS(WW)) to develop and evaluate two sets of 1RS/1RS(WW) near isogenic lines (NILs). Field trials showed that standard 1RS lines had significantly higher yield and better canopy water status than the 1RS(WW) NILs in both well-watered and water-stressed environments. We intercrossed the 1RS and 1RS(WW) lines and generated two additional NILs, one carrying the distal (1RS(RW)) and the other carrying the proximal (1RS(WR)) wheat segment. Lines not carrying the distal wheat region (1RS and 1RS(WR)) showed significant improvements in grain yield and canopy water status compared to NILs carrying the distal wheat segment (1RS(WW) and 1RS(RW)), indicating that the 1RS region replaced by the distal wheat segment carries the beneficial allele(s). NILs without the distal wheat segment also showed higher carbon isotope discrimination and increased stomatal conductance, suggesting that these plants had improved access to water. The 1RS(WW), 1RS(WR) and 1RS(RW) NILs have been deposited in the National Small Grains Collection.

Genome-wide diversity analysis suggests divergence among Upper Guinea and the Dahomey Gap populations of the Sisrè berry (Syn: miracle fruit) plant (Synsepalum dulcificum [Schumach. & Thonn.] Daniell) in West Africa.

Although Synsepalum dulcificum is viewed as one of the most economically promising orphan tree crops worldwide, its genetic improvement and sustainable conservation are hindered by a lack of understanding of its evolutionary history and current population structure. Here, we report for the first time the application of genome-wide single nucleotide polymorphism genotyping to a diverse panel of S. dulcificum accessions to depict the genetic diversity and population structure of the species in the Dahomey Gap (DG) and Upper Guinea (UG) regions to infer its evolutionary history. Our findings suggest low overall genetic diversity but strong population divergence within the species. Neighbor-joining analysis detected two genetic groups in the UG and DG regions, while STRUCTURE distinguished three genetic groups, corresponding to the UG, Western DG, and Central DG regions. Application of Monmonier's algorithm revealed the existence of a barrier disrupting connectivity between the UG and DG groups. The Western DG group consistently exhibited the highest levels of nucleotide and haplotype diversities, while that of the Central DG exhibited the lowest. Analyses of Tajima's D, Fu's Fs, and Achaz Y* statistics suggest that while both UG and Central DG groups likely experienced recent expansions, the Western DG group is at equilibrium. These findings suggest a geographical structuring of genetic variation which supports the conclusion of differential evolutionary histories among West African groups of S. dulcificum. These results provide foundational insights to guide informed breeding population development and design sustainable conservation strategies for this species.

'Systems approach' plant breeding illustrated by trees.

The linkage in new and creative ways of existing plant breeding methods responsive to different global trends and values provides a 'systems approach' to address a broad set of global production challenges more effectively. Here, we illustrate such an approach through its application to trees, chosen because of their extensive diversity in features, uses, users, production contexts, and domestication pathways. We coin the resulting strategy 'tree diversity breeding' and consider it with reference to trends and values related to participation, environment, biotechnology, and markets as examples. Features of the approach for trees are applicable to plant breeding more widely, as we seek to address complex problems through strategic biodiversity use.

Georeferenced phylogenetic analysis of a global collection of wild and cultivated Citrullus species.

The geographical origin of watermelon (Citrullus lanatus) remains debated. While a first hypothesis suggests the center of origin to be West Africa, where the endemic sister species C. mucosospermus thrives, a second hypothesis suggests northeastern Africa where the white-fleshed Sudanese Kordophan melon is cultivated. In this study, we infer biogeographical and haplotype genealogy for C. lanatus, C. mucosospermus, C. amarus, and C. colocynthis using noncoding cpDNA sequences (trnT-trnL and ndhF-rpl32 regions) from a global collection of 135 accessions. In total, we identified 38 haplotypes in C. lanatus, C. mucosospermus, C. amarus, and C. colocynthis; of these, 21 were found in Africa and 17 appear endemic to the continent. The least diverse species was C. mucosospermus (5 haplotypes) and the most diverse was C. colocynthis (16 haplotypes). Some haplotypes of C. mucosospermus were nearly exclusive to West Africa, and C. lanatus and C. mucosospermus shared haplotypes that were distinct from those of both C. amarus and C. colocynthis. The results support previous findings that revealed C. mucosospermus to be the closest relative to C. lanatus (including subsp. cordophanus). West Africa, as a center of endemism of C. mucosospermus, is an area of interest in the search of the origin of C. lanatus. This calls for further historical and phylogeographical investigations and wider collection of samples in West and northeastern Africa.

The Independent Domestication of Timopheev's Wheat: Insights from Haplotype Analysis of the Brittle rachis 1 (BTR1-A) Gene.

Triticum turgidum and T. timopheevii are two tetraploid wheat species sharing T. urartu as a common ancestor, and domesticated accessions from both of these allopolyploids exhibit nonbrittle rachis (i.e., nonshattering spikes). We previously described the loss-of-function mutations in the Brittle Rachis 1 genes BTR1-A and BTR1-B in the A and B subgenomes, respectively, that are responsible for this most visible domestication trait in T. turgidum. Resequencing of a large panel of wild and domesticated T. turgidum accessions subsequently led to the identification of the two progenitor haplotypes of the btr1-A and btr1-B domesticated alleles. Here, we extended the haplotype analysis to other T. turgidum subspecies and to the BTR1 homologues in the related T. timopheevii species. Our results showed that all the domesticated wheat subspecies within T. turgidum share common BTR1-A and BTR1-B haplotypes, confirming their common origin. In T. timopheevii, however, we identified a novel loss-of-function btr1-A allele underlying a partially brittle spike phenotype. This novel recessive allele appeared fixed within the pool of domesticated Timopheev's wheat but was also carried by one wild timopheevii accession exhibiting partial brittleness. The promoter region for BTR1-B could not be amplified in any T. timopheevii accessions with any T. turgidum primer combination, exemplifying the gene-level distance between the two species. Altogether, our results support the concept of independent domestication processes for the two polyploid, wheat-related species.

Genomic Resources to Guide Improvement of the Shea Tree.

A defining component of agroforestry parklands across Sahelo-Sudanian Africa (SSA), the shea tree (Vitellaria paradoxa) is central to sustaining local livelihoods and the farming environments of rural communities. Despite its economic and cultural value, however, not to mention the ecological roles it plays as a dominant parkland species, shea remains semi-domesticated with virtually no history of systematic genetic improvement. In truth, shea's extended juvenile period makes traditional breeding approaches untenable; but the opportunity for genome-assisted breeding is immense, provided the foundational resources are available. Here we report the development and public release of such resources. Using the FALCON-Phase workflow, 162.6 Gb of long-read PacBio sequence data were assembled into a 658.7 Mbp, chromosome-scale reference genome annotated with 38,505 coding genes. Whole genome duplication (WGD) analysis based on this gene space revealed clear signatures of two ancient WGD events in shea's evolutionary past, one prior to the Astrid-Rosid divergence (116-126 Mya) and the other at the root of the order Ericales (65-90 Mya). In a first genome-wide look at the suite of fatty acid (FA) biosynthesis genes that likely govern stearin content, the primary determinant of shea butter quality, relatively high copy numbers of six key enzymes were found (KASI, KASIII, FATB, FAD2, FAD3, and FAX2), some likely originating in shea's more recent WGD event. To help translate these findings into practical tools for characterization, selection, and genome-wide association studies (GWAS), resequencing data from a shea diversity panel was used to develop a database of more than 3.5 million functionally annotated, physically anchored SNPs. Two smaller, more curated sets of suggested SNPs, one for GWAS (104,211 SNPs) and the other targeting FA biosynthesis genes (90 SNPs), are also presented. With these resources, the hope is to support national programs across the shea belt in the strategic, genome-enabled conservation and long-term improvement of the shea tree for SSA.

Wheat domestication in light of haplotype analyses of the Brittle rachis 1 genes (BTR1-A and BTR1-B).

Wheat domestication was a milestone in the rise of agrarian societies in the Fertile Crescent. As opposed to the freely dispersing seeds of its tetraploid progenitor wild emmer, the hallmark trait of domesticated wheat is intact, harvestable spikes. During domestication, wheat acquired recessive loss-of-function mutations in the Brittle Rachis 1 genes, both in the A genome (BTR1-A) and B genome (BTR1-B). In this study, we probe the geographical provenances of these mutations via haplotype analyses of a collection of wild and domesticated accessions. Our results show that the precursor of the domesticated haplotype of BTR1-A was detected in 32% of the wild accessions gathered throughout the Levant, from central Israel to central Turkey. In contrast, the precursor of the domesticated haplotype of BTR1-B, which carries a distinct 11 bp deletion in the promoter region, was found in only 10% of the tested wild accessions, all from the Southern Levant. Moreover, we identified of a single wild emmer accession in Southern Levant that carries the progenitor haplotypes for both BTR1-A and BTR1-B genes. These observations suggest that at least part of the emmer domestication process occurred in Southern Levant, contrary to the widely held view that the northern part of the Fertile Crescent was the center of wheat domestication.

Genome Based Meta-QTL Analysis of Grain Weight in Tetraploid Wheat Identifies Rare Alleles of GRF4 Associated with Larger Grains.

The domestication and subsequent genetic improvement of wheat led to the development of large-seeded cultivated wheat species relative to their smaller-seeded wild progenitors. While increased grain weight (GW) continues to be an important goal of many wheat breeding programs, few genes underlying this trait have been identified despite an abundance of studies reporting quantitative trait loci (QTL) for GW. Here we perform a QTL analysis for GW using a population of recombinant inbred lines (RILs) derived from the cross between wild emmer wheat accession 'Zavitan' and durum wheat variety 'Svevo'. Identified QTLs in this population were anchored to the recent Zavitan reference genome, along with previously published QTLs for GW in tetraploid wheat. This genome-based, meta-QTL analysis enabled the identification of a locus on chromosome 6A whose introgression from wild wheat positively affects GW. The locus was validated using an introgression line carrying the 6A GW QTL region from Zavitan in a Svevo background, resulting in >8% increase in GW compared to Svevo. Using the reference sequence for the 6A QTL region, we identified a wheat ortholog to OsGRF4, a rice gene previously associated with GW. The coding sequence of this gene (TtGRF4-A) contains four single nucleotide polymorphisms (SNPs) between Zavitan and Svevo, one of which reveals the Zavitan allele to be rare in a core collection of wild emmer and completely absent from the domesticated emmer genepool. Similarly, another wild emmer accession (G18-16) was found to carry a rare allele of TtGRF4-A that also positively affects GW and is characterized by a unique SNP absent from the entire core collection. These results exemplify the rich genetic diversity of wild wheat, posit TtGRF4-A as a candidate gene underlying the 6A GW QTL, and suggest that the natural Zavitan and G18-16 alleles of TtGRF4-A have potential to increase wheat yields in breeding programs.

A roadmap for breeding orphan leafy vegetable species: a case study of Gynandropsis gynandra (Cleomaceae).

Despite an increasing awareness of the potential of "orphan" or unimproved crops to contribute to food security and enhanced livelihoods for farmers, coordinated research agendas to facilitate production and use of orphan crops by local communities are generally lacking. We provide an overview of the current knowledge on leafy vegetables with a focus on Gynandropsis gynandra, a highly nutritious species used in Africa and Asia, and highlight general and species-specific guidelines for participatory, genomics-assisted breeding of orphan crops. Key steps in genome-enabled orphan leafy vegetables improvement are identified and discussed in the context of Gynandropsis gynandra breeding, including: (1) germplasm collection and management; (2) product target definition and refinement; (3) characterization of the genetic control of key traits; (4) design of the 'process' for cultivar development; (5) integration of genomic data to optimize that 'process'; (6) multi-environmental participatory testing and end-user evaluation; and (7) crop value chain development. The review discusses each step in detail, with emphasis on improving leaf yield, phytonutrient content, organoleptic quality, resistance to biotic and abiotic stresses and post-harvest management.

GBS-Based Deconvolution of the Surviving North American Collection of Cold-Hardy Kiwifruit (Actinidia spp.) Germplasm.

Plant germplasm collections can be invaluable resources to plant breeders, provided they are well-characterized. After 140 years of acquisition and curation efforts by a wide and largely non-coordinated array of private and institutional actors, the current US collection of cold-hardy kiwifruit (Actinidia spp.) is rife with misclassifications, misnomers, and mix-ups. To facilitate the systematic improvement and resource-efficient curation of these species of long-recognized horticultural potential, we used genotyping-by-sequencing (GBS) data to deconvolute this historic collection. Evaluation of a total of 138 accessions (103 A. arguta, 28 A. kolomikta, and 7 A. polygama) with an interspecific set of 1,040 high-quality SNPs resulted in clear resolution of the three species. Intraspecific analysis (2,964 SNPs) within A. arguta revealed a significant level of redundancy (41.7%; only 60 unique genotypes out of 103 analyzed) and a sub-population structure reflecting likely geographic provenance, phenotypic classes, and hybrid pedigree. For A. kolomikta (3,425 SNPs), the level of accession redundancy was even higher (53.6%; 13 unique genotypes out of 28 analyzed); but no sub-structure was detected. Numerous instances were discovered of distinct genotypes sharing a common name, different names assigned to the same genotype, mistaken species assignments, and incorrect gender records, all critical information for both breeders and curators. In terms of method, this study demonstrates the practical and cost-effective use of GBS data to characterize plant genetic resources, despite ploidy differences and the lack of reference genomes. With the recent prohibition on further imports of Actinidia plant material into the country and with the active eradication of historic vines looming, this analysis of the US cold-hardy kiwifruit germplasm collection provides a timely assessment of the genetic resource base of an emerging, high-value specialty crop.

Early fruiting in Synsepalum dulcificum (Schumach. & Thonn.) Daniell juveniles induced by water and inorganic nutrient management.

Background. The miracle plant, Synsepalum dulcificum (Schumach. & Thonn.) Daniell is a native African orphan crop species that has recently received increased attention due to its promise as a sweetener and source of antioxidants in both the food and pharmaceutical industries. However, a major obstacle to the species' widespread utilization is its relatively slow growth rate and prolonged juvenile period. Method. In this study, we tested twelve treatments made up of various watering regimes and exogenous nutrient application (nitrogen, phosphorus and potassium, at varying dosages) on the relative survival, growth, and reproductive development of 15-months-old S. dulcificum juveniles. Results. While the plants survived under most tested growing conditions, nitrogen application at doses higher than 1.5 g [seedling] -1 was found to be highly detrimental, reducing survival to 0%. The treatment was found to affect all growth traits, and juveniles that received a combination of nitrogen, phosphorus, and potassium (each at a rate of 1.5 g [seedling] -1), in addition to daily watering, exhibited the most vegetative growth. The simple daily provision of adequate water was found to greatly accelerate the transition to reproductive maturity in the species (from >36 months to an average of 23 months), whereas nutrient application affected the length of the reproductive phase within a season, as well as the fruiting intensity. Conclusions. This study highlights the beneficial effect of water supply and fertilization on both vegetative and reproductive growth in S. dulcificum. Water supply appeared to be the most important factor unlocking flowering in the species, while the combination of nitrogen, phosphorus and potassium at the dose of 1.5 g (for all) consistently exhibited the highest performance for all growth and yield traits. These findings will help intensify S. dulcificum's breeding and horticultural development.