FruitPhenoBox - a device for rapid and automated fruit phenotyping of small sample sizes.

BACKGROUND: Fruit appearance of apple (Malus domestica Borkh.) is accession-specific and one of the main criteria for consumer choice. Consequently, fruit appearance is an important selection criterion in the breeding of new cultivars. It is also used for the description of older varieties or landraces. In commercial apple production, sorting devices are used to classify large numbers of fruit from a few cultivars. In contrast, the description of fruit from germplasm collections or breeding programs is based on only a few fruit from many accessions and is mostly performed visually by pomology experts. Such visual ratings are laborious, often difficult to compare and remain subjective. RESULTS: Here we report on a morphometric device, the FruitPhenoBox, for automated fruit weighing and appearance description using computer-based analysis of five images per fruit. Recording of approximately 100 fruit from each of 15 apple cultivars using the FruitPhenoBox was rapid, with an average handling and recording time of less than eleven seconds per fruit. Comparison of fruit images from the 15 apple cultivars identified significant differences in shape index, fruit width, height and weight. Fruit shape was characteristic for each cultivar, while fruit color showed larger variation within sample sets. Assessing a subset of 20 randomly selected fruit per cultivar, fruit height, width and weight were described with a relative margin of error of 2.6%, 2.2%, and 6.2%, respectively, calculated from the mean value of all available fruit. CONCLUSIONS: The FruitPhenoBox allows for the rapid and consistent description of fruit appearance from individual apple accessions. By relating the relative margin of error for fruit width, height and weight description with different sample sizes, it was possible to determine an appropriate fruit sample size to efficiently and accurately describe the recorded traits. Therefore, the FruitPhenoBox is a useful tool for breeding and the description of apple germplasm collections.

An improved chromosome-level genome assembly of perennial ryegrass (Lolium perenne L.).

This work is an update and extension of the previously published article "Ultralong Oxford Nanopore Reads Enable the Development of a Reference-Grade Perennial Ryegrass Genome Assembly" by Frei et al. The published genome assembly of the doubled haploid perennial ryegrass (Lolium perenne L.) genotype Kyuss (Kyuss v1.0) marked a milestone for forage grass research and breeding. However, order and orientation errors may exist in the pseudo-chromosomes of Kyuss, since barley (Hordeum vulgare L.), which diverged 30 million years ago from perennial ryegrass, was used as the reference to scaffold Kyuss. To correct for structural errors possibly present in the published Kyuss assembly, we de novo assembled the genome again and generated 50-fold coverage high-throughput chromosome conformation capture (Hi-C) data to assist pseudo-chromosome construction. The resulting new chromosome-level assembly Kyuss v2.0 showed improved quality with high contiguity (contig N50 = 120 Mb), high completeness (total BUSCO score = 99%), high base-level accuracy (QV = 50), and correct pseudo-chromosome structure (validated by Hi-C contact map). This new assembly will serve as a better reference genome for Lolium spp. and greatly benefit the forage and turf grass research community.

The genetic basis of apple shape and size unraveled by digital phenotyping.

Great diversity of shape, size, and skin color is observed among the fruits of different apple genotypes. These traits are critical for consumers and therefore interesting targets for breeding new apple varieties. However, they are difficult to phenotype and their genetic basis, especially for fruit shape and ground color, is largely unknown. We used the FruitPhenoBox to digitally phenotype 525 genotypes of the apple reference population (apple REFPOP) genotyped for 303,148 single nucleotide polymorphism (SNP) markers. From the apple images, 573 highly heritable features describing fruit shape and size as well as 17 highly heritable features for fruit skin color were extracted to explore genotype-phenotype relationships. Out of these features, seven principal components (PCs) and 16 features with the Pearson's correlation r < 0.75 (selected features) were chosen to carry out genome-wide association studies (GWAS) for fruit shape and size. Four PCs and eight selected features were used in GWAS for fruit skin color. In total, 69 SNPs scattered over all 17 apple chromosomes were significantly associated with round, conical, cylindrical, or symmetric fruit shapes and fruit size. Novel associations with major effect on round or conical fruit shapes and fruit size were identified on chromosomes 1 and 2. Additionally, 16 SNPs associated with PCs and selected features related to red overcolor as well as green and yellow ground color were found on eight chromosomes. The identified associations can be used to advance marker-assisted selection in apple fruit breeding to systematically select for desired fruit appearance.

High-resolution bulked segregant analysis enables candidate gene identification for bacterial wilt resistance in Italian ryegrass (Lolium multiflorum Lam.).

Bacterial wilt, caused by Xanthomonas translucens pv. graminis (Xtg), is a serious disease of economically important forage grasses, including Italian ryegrass (Lolium multiflorum Lam.). A major QTL for resistance to Xtg was previously identified, but the precise location as well as the genetic factors underlying the resistance are yet to be determined. To this end, we applied a bulked segregant analysis (BSA) approach, using whole-genome deep sequencing of pools of the most resistant and most susceptible individuals of a large (n = 7484) biparental F2 population segregating for resistance to Xtg. Using chromosome-level genome assemblies as references, we were able to define a ~300 kb region highly associated with resistance on pseudo-chromosome 4. Further investigation of this region revealed multiple genes with a known role in disease resistance, including genes encoding for Pik2-like disease resistance proteins, cysteine-rich kinases, and RGA4- and RGA5-like disease resistance proteins. Investigation of allele frequencies in the pools and comparative genome analysis in the grandparents of the F2 population revealed that some of these genes contain variants with allele frequencies that correspond to the expected heterozygosity in the resistant grandparent. This study emphasizes the efficacy of combining BSA studies in very large populations with whole genome deep sequencing and high-quality genome assemblies to pinpoint regions associated with a binary trait of interest and accurately define a small set of candidate genes. Furthermore, markers identified in this region hold significant potential for marker-assisted breeding strategies to breed resistance to Xtg in Italian ryegrass cultivars more efficiently.

Linking photosynthesis and yield reveals a strategy to improve light use efficiency in a climbing bean breeding population.

Photosynthesis drives plant physiology, biomass accumulation, and yield. Photosynthetic efficiency, specifically the operating efficiency of PSII (Fq'/Fm'), is highly responsive to actual growth conditions, especially to fluctuating photosynthetic photon fluence rate (PPFR). Under field conditions, plants constantly balance energy uptake to optimize growth. The dynamic regulation complicates the quantification of cumulative photochemical energy uptake based on the intercepted solar energy, its transduction into biomass, and the identification of efficient breeding lines. Here, we show significant effects on biomass related to genetic variation in photosynthetic efficiency of 178 climbing bean (Phaseolus vulgaris L.) lines. Under fluctuating conditions, the Fq'/Fm' was monitored throughout the growing period using hand-held and automated chlorophyll fluorescence phenotyping. The seasonal response of Fq'/Fm' to PPFR (ResponseG:PPFR) achieved significant correlations with biomass and yield, ranging from 0.33 to 0.35 and from 0.22 to 0.31 in two glasshouse and three field trials, respectively. Phenomic yield prediction outperformed genomic predictions for new environments in four trials under different growing conditions. Investigating genetic control over photosynthesis, one single nucleotide polymorphism (Chr09_37766289_13052) on chromosome 9 was significantly associated with ResponseG:PPFR in proximity to a candidate gene controlling chloroplast thylakoid formation. In conclusion, photosynthetic screening facilitates and accelerates selection for high yield potential.

High genomic plasticity and unique features of Xanthomonas translucens pv. graminis revealed through comparative analysis of complete genome sequences.

BACKGROUND: Xanthomonas translucens pv. graminis (Xtg) is a major bacterial pathogen of economically important forage grasses, causing severe yield losses. So far, genomic resources for this pathovar consisted mostly of draft genome sequences, and only one complete genome sequence was available, preventing comprehensive comparative genomic analyses. Such comparative analyses are essential in understanding the mechanisms involved in the virulence of pathogens and to identify virulence factors involved in pathogenicity. RESULTS: In this study, we produced high-quality, complete genome sequences of four strains of Xtg, complementing the recently obtained complete genome sequence of the Xtg pathotype strain. These genomic resources allowed for a comprehensive comparative analysis, which revealed a high genomic plasticity with many chromosomal rearrangements, although the strains were highly related. A high number of transposases were exclusively found in Xtg and corresponded to 413 to 457 insertion/excision transposable elements per strain. These mobile genetic elements are likely to be involved in the observed genomic plasticity and may play an important role in the adaptation of Xtg. The pathovar was found to lack a type IV secretion system, and it possessed the smallest set of type III effectors in the species. However, three XopE and XopX family effectors were found, while in the other pathovars of the species two or less were present. Additional genes that were specific to the pathovar were identified, including a unique set of minor pilins of the type IV pilus, 17 TonB-dependent receptors (TBDRs), and 11 plant cell wall degradative enzymes. CONCLUSION: These results suggest a high adaptability of Xtg, conferred by the abundance of mobile genetic elements, which could play a crucial role in pathogen adaptation. The large amount of such elements in Xtg compared to other pathovars of the species could, at least partially, explain its high virulence and broad host range. Conserved features that were specific to Xtg were identified, and further investigation will help to determine genes that are essential to pathogenicity and host adaptation of Xtg.

ChromaX: a fast and scalable breeding program simulator.

SUMMARY: ChromaX is a Python library that enables the simulation of genetic recombination, genomic estimated breeding value calculations, and selection processes. By utilizing GPU processing, it can perform these simulations up to two orders of magnitude faster than existing tools with standard hardware. This offers breeders and scientists new opportunities to simulate genetic gain and optimize breeding schemes. AVAILABILITY AND IMPLEMENTATION: The documentation is available at https://chromax.readthedocs.io. The code is available at https://github.com/kora-labs/chromax.

Comparative and population genomics of buckwheat species reveal key determinants of flavor and fertility.

Common buckwheat (Fagopyrum esculentum) is an ancient crop with a world-wide distribution. Due to its excellent nutritional quality and high economic and ecological value, common buckwheat is becoming increasingly important throughout the world. The availability of a high-quality reference genome sequence and population genomic data will accelerate the breeding of common buckwheat, but the high heterozygosity due to the outcrossing nature has greatly hindered the genome assembly. Here we report the assembly of a chromosome-scale high-quality reference genome of F. esculentum var. homotropicum, a homozygous self-pollinating variant of common buckwheat. Comparative genomics revealed that two cultivated buckwheat species, common buckwheat (F. esculentum) and Tartary buckwheat (F. tataricum), underwent metabolomic divergence and ecotype differentiation. The expansion of several gene families in common buckwheat, including FhFAR genes, is associated with its wider distribution than Tartary buckwheat. Copy number variation of genes involved in the metabolism of flavonoids is associated with the difference of rutin content between common and Tartary buckwheat. Furthermore, we present a comprehensive atlas of genomic variation based on whole-genome resequencing of 572 accessions of common buckwheat. Population and evolutionary genomics reveal genetic variation associated with environmental adaptability and floral development between Chinese and non-Chinese cultivated groups. Genome-wide association analyses of multi-year agronomic traits with the content of flavonoids revealed that Fh05G014970 is a potential major regulator of flowering period, a key agronomic trait controlling the yield of outcrossing crops, and that Fh06G015130 is a crucial gene underlying flavor-associated flavonoids. Intriguingly, we found that the gene translocation and sequence variation of FhS-ELF3 contribute to the homomorphic self-compatibility of common buckwheat. Collectively, our results elucidate the genetic basis of speciation, ecological adaptation, fertility, and unique flavor of common buckwheat, and provide new resources for future genomics-assisted breeding of this economically important crop.

Genome-wide association study of lipase and esterase in wholegrain wheat flour (Triticum aestivum L.).

Lipase activity is one of the main causes of the lipid rancidity in wholegrain wheat flour, leading to its short shelf life. Genetically diverse wheat germplasm offers potential for the selection of wheat cultivars with low lipase activity for stable wholegrain end use. This study evaluated 300 European wheat cultivars harvested in 2015 and 2016 on the genetic association of lipase and esterase activities in wholegrain wheat flour. Esterase and lipase activities in wholegrain flour were measured photometrically with p-nitrophenyl butyrate and p-nitrophenyl palmitate as substrates, respectively. Both enzyme activities showed wide ranges among all cultivars within each year, with differences up to 2.5-fold. The two years showed low correlations between each other, indicating a large environmental impact on the enzyme activities. Cultivars 'Julius' and 'Bueno' were suggested to be better suited for stable wholegrain products, as they had consistently low esterase and lipase activities compared to the other cultivars. A genome-wide association study revealed associations with single nucleotide polymorphisms in genes located on the high-quality wheat genome sequence of the International Wheat Genome Sequencing Consortium. Eight and four candidate genes were tentatively proposed to be associated to esterase and lipase activity, respectively, in wholegrain flour. Our work shows esterase and lipase activities from a new perspective, that combines reverse genetics to understand the underlying causes. This study outlines the possibilities and limitations to improve lipid stability of wholegrain wheat by genomics-assisted breeding methods, thereby offering new opportunities to optimize the quality of wholegrain wheat flour and wholegrain products.

Both male and female meiosis contribute to non-Mendelian inheritance of parental chromosomes in interspecific plant hybrids (Lolium × Festuca).

Some interspecific plant hybrids show unequal transmission of chromosomes from parental genomes to the successive generations. It has been suggested that this is due to a differential behavior of parental chromosomes during meiosis. However, underlying mechanism is unknown. We analyzed chromosome composition of the F2 generation of Festuca × Lolium hybrids and reciprocal backcrosses to elucidate effects of male and female meiosis on the shift in parental genome composition. We studied male meiosis, including the attachment of chromosomes to the karyokinetic spindle and gene expression profiling of the kinetochore genes. We found that Lolium and Festuca homoeologues were transmitted differently to the F2 generation. Female meiosis led to the replacement of Festuca chromosomes by their Lolium counterparts. In male meiosis, Festuca univalents were attached less frequently to microtubules than Lolium univalents, lagged in divisions and formed micronuclei, which were subsequently eliminated. Genome sequence analysis revealed a number of non-synonymous mutations between copies of the kinetochore genes from Festuca and Lolium genomes. Furthermore, we found that outer kinetochore proteins NDC80 and NNF1 were exclusively expressed from the Lolium allele. We hypothesize that silencing of Festuca alleles results in improper attachment of Festuca chromosomes to karyokinetic spindle and subsequently their gradual elimination.

Field study of the fire-blight-resistant cisgenic apple line C44.4.146.

Cisgenesis, the genetic modification of a plant with genes from a sexually compatible plant, was used to confer fire blight resistance to the cultivar 'Gala Galaxy' by amendment of the resistance gene FB_MR5, resulting in the line C44.4.146. To verify whether cisgenesis changed other tree-, flower- or fruit-related traits, a 5-year field trial was conducted with trees of C44.4.146 and multiple control genotypes, including members of the 'Gala' sports group. None of the 44 investigated tree-, flower- or fruit-related traits significantly differed between C44.4.146 and at least one of the control genotypes in all observation years. However, fruits of C44.4.146 and its wild-type 'Gala Galaxy' from tissue culture were paler in color than fruits of 'Gala Galaxy' that had not undergone tissue culture. There was no significant and consistently detected difference in the fruit flesh and peel metabolome of C44.4.146 compared with the control genotypes. Finally, the disease resistance of C44.4.146 was confirmed also when the fire blight pathogen was inoculated through the flowers. We conclude that the use of cisgenesis to confer fire blight resistance to 'Gala Galaxy' in C44.4.146 did not have unintended effects, and that the in vitro establishment of 'Gala Galaxy' had a greater effect on C44.4.146 properties than its generation applying cisgenesis.

Genetic analysis of resistance to bean leaf crumple virus identifies a candidate LRR-RLK gene.

Bean leaf crumple virus (BLCrV) is a novel begomovirus (family Geminiviridae, genus Begomovirus) infecting common bean (Phaseolus vulgaris L.), threatening bean production in Latin America. Genetic resistance is required to ensure yield stability and reduce the use of insecticides, yet the available resistance sources are limited. In this study, three common bean populations containing a total of 558 genotypes were evaluated in different yield and BLCrV resistance trials under natural infection in the field. A genome-wide association study identified the locus BLC7.1 on chromosome Pv07 at 3.31 Mbp, explaining 8 to 16% of the phenotypic variation for BLCrV resistance. In comparison, whole-genome regression models explained 51 to 78% of the variation and identified the same region on Pv07 to confer resistance. The most significantly associated markers were located within the gene model Phvul.007G040400, which encodes a leucine-rich repeat receptor-like kinase subfamily III member and is likely to be involved in the innate immune response against the virus. The allelic diversity within this gene revealed five different haplotype groups, one of which was significantly associated with BLCrV resistance. As the same genome region was previously reported to be associated with resistance against other geminiviruses affecting common bean, our study highlights the role of previous breeding efforts for virus resistance in the accumulation of positive alleles against newly emerging viruses. In addition, we provide novel diagnostic single-nucleotide polymorphism markers for marker-assisted selection to exploit BLC7.1 for breeding against geminivirus diseases in one of the most important food crops worldwide.

Fine-Mapping and Comparative Genomic Analysis Reveal the Gene Composition at the S and Z Self-incompatibility Loci in Grasses.

Self-incompatibility (SI) is a genetic mechanism of hermaphroditic plants to prevent inbreeding after self-pollination. Allogamous Poaceae species exhibit a unique gametophytic SI system controlled by two multi-allelic and independent loci, S and Z. Despite intense research efforts in the last decades, the genes that determine the initial recognition mechanism are yet to be identified. Here, we report the fine-mapping of the Z-locus in perennial ryegrass (Lolium perenne L.) and provide evidence that the pollen and stigma components are determined by two genes encoding DUF247 domain proteins (ZDUF247-I and ZDUF247-II) and the gene sZ, respectively. The pollen and stigma determinants are located side-by-side and were genetically linked in 10,245 individuals of two independent mapping populations segregating for Z. Moreover, they exhibited high allelic diversity as well as tissue-specific gene expression, matching the expected characteristics of SI determinants known from other systems. Revisiting the S-locus using the latest high-quality whole-genome assemblies revealed a similar gene composition and structure as found for Z, supporting the hypothesis of a duplicated origin of the two-locus SI system of grasses. Ultimately, comparative genomic analyses across a wide range of self-compatible and self-incompatible Poaceae species revealed that the absence of a functional copy of at least one of the six putative SI determinants is accompanied by a self-compatible phenotype. Our study provides new insights into the origin and evolution of the unique gametophytic SI system in one of the largest and economically most important plant families.

Phenotypic variation and quantitative trait loci for resistance to southern anthracnose and clover rot in red clover.

KEY MESSAGE: High variability for and candidate loci associated with resistance to southern anthracnose and clover rot in a worldwide collection of red clover provide a first basis for genomics-assisted breeding. Red clover (Trifolium pratense L.) is an important forage legume of temperate regions, particularly valued for its high yield potential and its high forage quality. Despite substantial breeding progress during the last decades, continuous improvement of cultivars is crucial to ensure yield stability in view of newly emerging diseases or changing climatic conditions. The high amount of genetic diversity present in red clover ecotypes, landraces, and cultivars provides an invaluable, but often unexploited resource for the improvement of key traits such as yield, quality, and resistance to biotic and abiotic stresses. A collection of 397 red clover accessions was genotyped using a pooled genotyping-by-sequencing approach with 200 plants per accession. Resistance to the two most pertinent diseases in red clover production, southern anthracnose caused by Colletotrichum trifolii, and clover rot caused by Sclerotinia trifoliorum, was assessed using spray inoculation. The mean survival rate for southern anthracnose was 22.9% and the mean resistance index for clover rot was 34.0%. Genome-wide association analysis revealed several loci significantly associated with resistance to southern anthracnose and clover rot. Most of these loci are in coding regions. One quantitative trait locus (QTL) on chromosome 1 explained 16.8% of the variation in resistance to southern anthracnose. For clover rot resistance we found eight QTL, explaining together 80.2% of the total phenotypic variation. The SNPs associated with these QTL provide a promising resource for marker-assisted selection in existing breeding programs, facilitating the development of novel cultivars with increased resistance against two devastating fungal diseases of red clover.

Callus Induction from Diverse Explants and Genotypes Enables Robust Transformation of Perennial Ryegrass (Lolium perenne L.).

Genetic transformation of perennial ryegrass (Lolium perenne L.) is critical for fundamental and translational research in this important grass species. It often relies on Agrobacterium-mediated transformation of callus tissue. However, callus induction is restricted to a few genotypes that respond well to tissue culture. Here, we report callus induction from different perennial ryegrass genotypes and explants, such as shoot tips, seeds, and anthers, which were transformed with several plasmids for functional genomics. ß-glucuronidase (GUS) histochemical staining showed the LmdsRNAbp promoter sequence was active in stigmas, spikelets, anthers, and leaves. We also transformed calli with plasmids allowing gene silencing and gene knock-out using RNA interference and CRISPR/Cas9, respectively, for which genotypic and phenotypic investigations are ongoing. Using 19 different constructs, 262 transgenic events were regenerated. Moreover, the protocol regenerated a doubled haploid transgenic event from anther-derived calli. This work provides a proof-of-concept method for expanding the range of genotypes amenable to transformation, thus, serving research and breeding initiatives to improve this important grass crop for forage and recreation.

No adverse dietary effect of a cisgenic fire blight resistant apple line on the non-target arthropods Drosophila melanogaster and Folsomia candida.

Genetic modification of apple cultivars through cisgenesis can introduce traits, such as disease resistance from wild relatives, quickly and without crossing. This approach was used to generate the cisgenic apple line C44.4.146, a 'Gala Galaxy' carrying the fire blight resistance gene FB_MR5. In contrast to traditionally bred apple cultivars, genetically modified (GM) plants need to undergo a regulatory risk assessment considering unintended effects before approval for commercial release. To determine potential unintended effects of C44.4.146, we assessed major leaf components and effects on the fitness of the decomposers Drosophila melanogaster (fruit fly) and Folsomia candida (collembolan), which were fed a diet amended with powdered apple leaf material. Leaf material of 'Gala Galaxy', several natural 'Gala' mutants, and the unrelated apple cultivar 'Ladina' were used for comparison. The genetic modification did not alter major leaf components and did not adversely affect survival, growth, or fecundity of the two decomposers. Consistent with previous studies with other GM crops, the differences between conventionally bred cultivars were greater than between the GM line and its non-GM wild type. These data provide a baseline for future risk assessments.

Low-Input High-Molecular-Weight DNA Extraction for Long-Read Sequencing From Plants of Diverse Families.

Long-read DNA sequencing technologies require high molecular weight (HMW) DNA of adequate purity and integrity, which can be difficult to isolate from plant material. Plant leaves usually contain high levels of carbohydrates and secondary metabolites that can impact DNA purity, affecting downstream applications. Several protocols and kits are available for HMW DNA extraction, but they usually require a high amount of input material and often lead to substantial DNA fragmentation, making sequencing suboptimal in terms of read length and data yield. We here describe a protocol for plant HMW DNA extraction from low input material (0.1 g) which is easy to follow and quick (2.5 h). This method successfully enabled us to extract HMW from four species from different families (Orchidaceae, Poaceae, Brassicaceae, Asteraceae). In the case of recalcitrant species, we show that an additional purification step is sufficient to deliver a clean DNA sample. We demonstrate the suitability of our protocol for long-read sequencing on the Oxford Nanopore Technologies PromethION® platform, with and without the use of a short fragment depletion kit.

Improving Association Studies and Genomic Predictions for Climbing Beans With Data From Bush Bean Populations.

Common bean (Phaseolus vulgaris L.) has two major origins of domestication, Andean and Mesoamerican, which contribute to the high diversity of growth type, pod and seed characteristics. The climbing growth habit is associated with increased days to flowering (DF), seed iron concentration (SdFe), nitrogen fixation, and yield. However, breeding efforts in climbing beans have been limited and independent from bush type beans. To advance climbing bean breeding, we carried out genome-wide association studies and genomic predictions using 1,869 common bean lines belonging to five breeding panels representing both gene pools and all growth types. The phenotypic data were collected from 17 field trials and were complemented with 16 previously published trials. Overall, 38 significant marker-trait associations were identified for growth habit, 14 for DF, 13 for 100 seed weight, three for SdFe, and one for yield. Except for DF, the results suggest a common genetic basis for traits across all panels and growth types. Seven QTL associated with growth habits were confirmed from earlier studies and four plausible candidate genes for SdFe and 100 seed weight were newly identified. Furthermore, the genomic prediction accuracy for SdFe and yield in climbing beans improved up to 8.8% when bush-type bean lines were included in the training population. In conclusion, a large population from different gene pools and growth types across multiple breeding panels increased the power of genomic analyses and provides a solid and diverse germplasm base for genetic improvement of common bean.

Genetic architecture and genomic predictive ability of apple quantitative traits across environments.

Implementation of genomic tools is desirable to increase the efficiency of apple breeding. Recently, the multi-environment apple reference population (apple REFPOP) proved useful for rediscovering loci, estimating genomic predictive ability, and studying genotype by environment interactions (G × E). So far, only two phenological traits were investigated using the apple REFPOP, although the population may be valuable when dissecting genetic architecture and reporting predictive abilities for additional key traits in apple breeding. Here we show contrasting genetic architecture and genomic predictive abilities for 30 quantitative traits across up to six European locations using the apple REFPOP. A total of 59 stable and 277 location-specific associations were found using GWAS, 69.2% of which are novel when compared with 41 reviewed publications. Average genomic predictive abilities of 0.18-0.88 were estimated using main-effect univariate, main-effect multivariate, multi-environment univariate, and multi-environment multivariate models. The G × E accounted for up to 24% of the phenotypic variability. This most comprehensive genomic study in apple in terms of trait-environment combinations provided knowledge of trait biology and prediction models that can be readily applied for marker-assisted or genomic selection, thus facilitating increased breeding efficiency.

A multispecies amplicon sequencing approach for genetic diversity assessments in grassland plant species.

Grasslands are widespread and economically relevant ecosystems at the basis of sustainable roughage production. Plant genetic diversity (PGD; i.e., within-species diversity) is related to many beneficial effects on the ecosystem functioning of grasslands. The monitoring of PGD in temperate grasslands is complicated by the multiplicity of species present and by a shortage of methods for large-scale assessments. However, the continuous advancement of high-throughput DNA sequencing approaches has improved the prospects of broad, multispecies PGD monitoring. Among them, amplicon sequencing stands out as a robust and cost-effective method. Here, we report a set of 12 multispecies primer pairs that can be used for high-throughput PGD assessments in multiple grassland plant species. The target loci were selected and tested in two phases: a "discovery phase" based on a sequence capture assay (611 nuclear loci assessed in 16 grassland plant species), which resulted in the selection of 11 loci; and a "validation phase", in which the selected loci were targeted and sequenced using multispecies primers in test populations of Dactylis glomerata L., Lolium perenne L., Festuca pratensis Huds., Trifolium pratense L. and T. repens L. The multispecies amplicons had nucleotide diversities per species from 5.19 × 10-3 to 1.29 × 10-2 , which is in the range of flowering-related genes but slightly lower than pathogen resistance genes. We conclude that the methodology, the DNA sequence resources, and the primer pairs reported in this study provide the basis for large-scale, multispecies PGD monitoring in grassland plants.