Genome sequencing of adapted diploid potato clones.

Cultivated potato is a vegetatively propagated crop, and most varieties are autotetraploid with high levels of heterozygosity. Reducing the ploidy and breeding potato at the diploid level can increase efficiency for genetic improvement including greater ease of introgression of diploid wild relatives and more efficient use of genomics and markers in selection. More recently, selfing of diploids for generation of inbred lines for F1 hybrid breeding has had a lot of attention in potato. The current study provides genomics resources for nine legacy non-inbred adapted diploid potato clones developed at Agriculture and Agri-Food Canada. De novo genome sequence assembly using 10× Genomics and Illumina sequencing technologies show the genome sizes ranged from 712 to 948 Mbp. Structural variation was identified by comparison to two references, the potato DMv6.1 genome and the phased RHv3 genome, and a k-mer based analysis of sequence reads showed the genome heterozygosity range of 1 to 9.04% between clones. A genome-wide approach was taken to scan 5 Mb bins to visualize patterns of heterozygous deleterious alleles. These were found dispersed throughout the genome including regions overlapping segregation distortions. Novel variants of the StCDF1 gene conferring earliness of tuberization were found among these clones, which all produce tubers under long days. The genomes will be useful tools for genome design for potato breeding.

Phased, chromosome-scale genome assemblies of tetraploid potato reveal a complex genome, transcriptome, and predicted proteome landscape underpinning genetic diversity.

Cultivated potato is a clonally propagated autotetraploid species with a highly heterogeneous genome. Phased assemblies of six cultivars including two chromosome-scale phased genome assemblies revealed extensive allelic diversity, including altered coding and transcript sequences, preferential allele expression, and structural variation that collectively result in a highly complex transcriptome and predicted proteome, which are distributed across the homologous chromosomes. Wild species contribute to the extensive allelic diversity in tetraploid cultivars, demonstrating ancestral introgressions predating modern breeding efforts. As a clonally propagated autotetraploid that undergoes limited meiosis, dysfunctional and deleterious alleles are not purged in tetraploid potato. Nearly a quarter of the loci bore mutations are predicted to have a high negative impact on protein function, complicating breeder's efforts to reduce genetic load. The StCDF1 locus controls maturity, and analysis of six tetraploid genomes revealed that 12 allelic variants of StCDF1 are correlated with maturity in a dosage-dependent manner. Knowledge of the complexity of the tetraploid potato genome with its rampant structural variation and embedded deleterious and dysfunctional alleles will be key not only to implementing precision breeding of tetraploid cultivars but also to the construction of homozygous, diploid potato germplasm containing favorable alleles to capitalize on heterosis in F1 hybrids.

Genomic consequences of apple improvement.

The apple (Malus domestica) is one of the world's most commercially important perennial crops and its improvement has been the focus of human effort for thousands of years. Here, we genetically characterise over 1000 apple accessions from the United States Department of Agriculture (USDA) germplasm collection using over 30,000 single-nucleotide polymorphisms (SNPs). We confirm the close genetic relationship between modern apple cultivars and their primary progenitor species, Malus sieversii from Central Asia, and find that cider apples derive more of their ancestry from the European crabapple, Malus sylvestris, than do dessert apples. We determine that most of the USDA collection is a large complex pedigree: over half of the collection is interconnected by a series of first-degree relationships. In addition, 15% of the accessions have a first-degree relationship with one of the top 8 cultivars produced in the USA. With the exception of 'Honeycrisp', the top 8 cultivars are interconnected to each other via pedigree relationships. The cultivars 'Golden Delicious' and 'Red Delicious' were found to have over 60 first-degree relatives, consistent with their repeated use by apple breeders. We detected a signature of intense selection for red skin and provide evidence that breeders also selected for increased firmness. Our results suggest that Americans are eating apples largely from a single family tree and that the apple's future improvement will benefit from increased exploitation of its tremendous natural genetic diversity.

Genome-Wide Association Studies in Apple Reveal Loci for Aroma Volatiles, Sugar Composition, and Harvest Date.

Understanding the genetic architecture of fruit quality traits is crucial to target breeding of apple ( L.) cultivars. We linked genotype and phenotype information by combining genotyping-by-sequencing (GBS) generated single nucleotide polymorphism (SNP) markers with fruit flavor volatile data, sugar and acid content, and historical trait data from a gene bank collection. Using gas chromatography-mass spectrometry (GC-MS) analysis of apple juice samples, we identified 49 fruit volatile organic compounds (VOCs). We found a very variable content of VOCs, especially for the esters, among 149 apple cultivars. We identified convincing associations for the acetate esters especially butyl acetate and hexyl acetate on chromosome 2 in a region of several alcohol acyl-transferases including AAT1. For sucrose content and for fructose and sucrose in percentage of total sugars, we revealed significant SNP associations. Here, we suggest a vacuolar invertase close to significant SNPs for this association as candidate gene. Harvest date was in strong SNP association with a NAC transcription factor gene and sequencing identified two haplotypes associated with harvest date. The study shows that SNP marker characterization of a gene bank collection can be successfully combined with new and historical trait data for association studies. Suggested candidate genes may contribute to an improved understanding of the genetic basis for important traits and simultaneously provide tools for targeted breeding using marker-assisted selection (MAS).

A Genome-Wide Association Study of Apple Quality and Scab Resistance.

The apple ( × Borkh.) is an economically and culturally important crop grown worldwide. Growers of this long-lived perennial must produce fruit of adequate quality while also combatting abiotic and biotic stress. Traditional apple breeding can take up to 20 yr from initial cross to commercial release, but genomics-assisted breeding can help accelerate this process. To advance genomics-assisted breeding in apple, we performed genome-wide association studies (GWAS) and genomic prediction in a collection of 172 apple accessions by linking over 55,000 single nucleotide polymorphisms (SNPs) with 10 phenotypes collected over 2 yr. Genome-wide association studies revealed several known loci for skin color, harvest date and firmness at harvest. Several significant GWAS associations were detected for resistance to a major fungal pathogen, apple scab ( [Cke.] Wint.), but we demonstrate that these hits likely represent a single ancestral source. Using genomic prediction, we show that most phenotypes are sufficiently predictable using genome-wide SNPs to be candidates for genomic selection. Finally, we detect a signal for firmness retention after storage on chromosome 10 and show that it may not stem from variation in , a gene repeatedly identified in bi-parental mapping studies and widely believed to underlie a major QTL for firmness on chromosome 10. We provide evidence that this major QTL is more likely due to variation in a neighboring ethylene response factor (ERF) gene. The present study showcases the superior mapping resolution of GWAS compared to bi-parental linkage mapping by identifying a novel candidate gene underlying a well-studied, major QTL involved in apple firmness.

Patterns of genomic and phenomic diversity in wine and table grapes.

Grapes are one of the most economically and culturally important crops worldwide, and they have been bred for both winemaking and fresh consumption. Here we evaluate patterns of diversity across 33 phenotypes collected over a 17-year period from 580 table and wine grape accessions that belong to one of the world's largest grape gene banks, the grape germplasm collection of the United States Department of Agriculture. We find that phenological events throughout the growing season are correlated, and quantify the marked difference in size between table and wine grapes. By pairing publicly available historical phenotype data with genome-wide polymorphism data, we identify large effect loci controlling traits that have been targeted during domestication and breeding, including hermaphroditism, lighter skin pigmentation and muscat aroma. Breeding for larger berries in table grapes was traditionally concentrated in geographic regions where Islam predominates and alcohol was prohibited, whereas wine grapes retained the ancestral smaller size that is more desirable for winemaking in predominantly Christian regions. We uncover a novel locus with a suggestive association with berry size that harbors a signature of positive selection for larger berries. Our results suggest that religious rules concerning alcohol consumption have had a marked impact on patterns of phenomic and genomic diversity in grapes.

Genome to Phenome Mapping in Apple Using Historical Data.

Apple ( X Borkh.) is one of the world's most valuable fruit crops. Its large size and long juvenile phase make it a particularly promising candidate for marker-assisted selection (MAS). However, advances in MAS in apple have been limited by a lack of phenotype and genotype data from sufficiently large samples. To establish genotype-phenotype relationships and advance MAS in apple, we extracted over 24,000 phenotype scores from the USDA-Germplasm Resources Information Network (GRIN) database and linked them with over 8000 single nucleotide polymorphisms (SNPs) from 689 apple accessions from the USDA apple germplasm collection clonally preserved in Geneva, NY. We find significant genetic differentiation between Old World and New World cultivars and demonstrate that the genetic structure of the domesticated apple also reflects the time required for ripening. A genome-wide association study (GWAS) of 36 phenotypes confirms the association between fruit color and the MYB1 locus, and we also report a novel association between the transcription factor, NAC18.1, and harvest date and fruit firmness. We demonstrate that harvest time and fruit size can be predicted with relatively high accuracies ( > 0.46) using genomic prediction. Rapid decay of linkage disequilibrium (LD) in apples means millions of SNPs may be required for well-powered GWAS. However, rapid LD decay also promises to enable extremely high resolution mapping of causal variants, which holds great potential for advancing MAS.

QTL analysis of soft scald in two apple populations.

The apple (Malus×domestica Borkh.) is one of the world's most widely grown and valuable fruit crops. With demand for apples year round, storability has emerged as an important consideration for apple breeding programs. Soft scald is a cold storage-related disorder that results in sunken, darkened tissue on the fruit surface. Apple breeders are keen to generate new cultivars that do not suffer from soft scald and can thus be marketed year round. Traditional breeding approaches are protracted and labor intensive, and therefore marker-assisted selection (MAS) is a valuable tool for breeders. To advance MAS for storage disorders in apple, we used genotyping-by-sequencing (GBS) to generate high-density genetic maps in two F1 apple populations, which were then used for quantitative trait locus (QTL) mapping of soft scald. In total, 900 million DNA sequence reads were generated, but after several data filtering steps, only 2% of reads were ultimately used to create two genetic maps that included 1918 and 2818 single-nucleotide polymorphisms. Two QTL associated with soft scald were identified in one of the bi-parental populations originating from parent 11W-12-11, an advanced breeding line. This study demonstrates the utility of next-generation DNA sequencing technologies for QTL mapping in F1 populations, and provides a basis for the advancement of MAS to improve storability of apples.

The Genetic Structure of Marijuana and Hemp.

Despite its cultivation as a source of food, fibre and medicine, and its global status as the most used illicit drug, the genus Cannabis has an inconclusive taxonomic organization and evolutionary history. Drug types of Cannabis (marijuana), which contain high amounts of the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC), are used for medical purposes and as a recreational drug. Hemp types are grown for the production of seed and fibre, and contain low amounts of THC. Two species or gene pools (C. sativa and C. indica) are widely used in describing the pedigree or appearance of cultivated Cannabis plants. Using 14,031 single-nucleotide polymorphisms (SNPs) genotyped in 81 marijuana and 43 hemp samples, we show that marijuana and hemp are significantly differentiated at a genome-wide level, demonstrating that the distinction between these populations is not limited to genes underlying THC production. We find a moderate correlation between the genetic structure of marijuana strains and their reported C. sativa and C. indica ancestry and show that marijuana strain names often do not reflect a meaningful genetic identity. We also provide evidence that hemp is genetically more similar to C. indica type marijuana than to C. sativa strains.

Genomics: a potential panacea for the perennial problem.

Perennial crops represent important fresh and processed food sources worldwide, but advancements in breeding perennials are often impeded due to their very nature. The perennial crops we rely on most for food take several years to reach production maturity and require large spaces to grow, which make breeding new cultivars costly compared with most annual crops. Because breeding perennials is inefficient and expensive, they are often grown in monocultures consisting of small numbers of elite cultivars that are vegetatively propagated for decades or even centuries. This practice puts many perennial crops at risk for calamity since they remain stationary in the face of evolving pest and disease pressures. Although there is tremendous genetic diversity available to them, perennial crop breeders often struggle to generate commercially successful cultivars in a timely and cost-effective manner because of the high costs of breeding. Moreover, consumers often expect the same cultivars to be available indefinitely, and there is often little or no incentive for growers and retailers to take the risk of adopting new cultivars. While genomics studies linking DNA variants to commercially important traits have been performed in diverse perennial crops, the translation of these studies into accelerated breeding of improved cultivars has been limited. Here we explain the "perennial problem" in detail and demonstrate how modern genomics tools can significantly improve the cost effectiveness of breeding perennial crops and thereby prevent crucial food sources from succumbing to the perils of perpetual propagation.

Fast and cost-effective genetic mapping in apple using next-generation sequencing.

Next-generation DNA sequencing (NGS) produces vast amounts of DNA sequence data, but it is not specifically designed to generate data suitable for genetic mapping. Recently developed DNA library preparation methods for NGS have helped solve this problem, however, by combining the use of reduced representation libraries with DNA sample barcoding to generate genome-wide genotype data from a common set of genetic markers across a large number of samples. Here we use such a method, called genotyping-by-sequencing (GBS), to produce a data set for genetic mapping in an F1 population of apples (Malus × domestica) segregating for skin color. We show that GBS produces a relatively large, but extremely sparse, genotype matrix: over 270,000 SNPs were discovered but most SNPs have too much missing data across samples to be useful for genetic mapping. After filtering for genotype quality and missing data, only 6% of the 85 million DNA sequence reads contributed to useful genotype calls. Despite this limitation, using existing software and a set of simple heuristics, we generated a final genotype matrix containing 3967 SNPs from 89 DNA samples from a single lane of Illumina HiSeq and used it to create a saturated genetic linkage map and to identify a known QTL underlying apple skin color. We therefore demonstrate that GBS is a cost-effective method for generating genome-wide SNP data suitable for genetic mapping in a highly diverse and heterozygous agricultural species. We anticipate future improvements to the GBS analysis pipeline presented here that will enhance the utility of next-generation DNA sequence data for the purposes of genetic mapping across diverse species.

SNP discovery and chromosome anchoring provide the first physically-anchored hexaploid oat map and reveal synteny with model species.

A physically anchored consensus map is foundational to modern genomics research; however, construction of such a map in oat (Avena sativa L., 2n = 6x = 42) has been hindered by the size and complexity of the genome, the scarcity of robust molecular markers, and the lack of aneuploid stocks. Resources developed in this study include a modified SNP discovery method for complex genomes, a diverse set of oat SNP markers, and a novel chromosome-deficient SNP anchoring strategy. These resources were applied to build the first complete, physically-anchored consensus map of hexaploid oat. Approximately 11,000 high-confidence in silico SNPs were discovered based on nine million inter-varietal sequence reads of genomic and cDNA origin. GoldenGate genotyping of 3,072 SNP assays yielded 1,311 robust markers, of which 985 were mapped in 390 recombinant-inbred lines from six bi-parental mapping populations ranging in size from 49 to 97 progeny. The consensus map included 985 SNPs and 68 previously-published markers, resolving 21 linkage groups with a total map distance of 1,838.8 cM. Consensus linkage groups were assigned to 21 chromosomes using SNP deletion analysis of chromosome-deficient monosomic hybrid stocks. Alignments with sequenced genomes of rice and Brachypodium provide evidence for extensive conservation of genomic regions, and renewed encouragement for orthology-based genomic discovery in this important hexaploid species. These results also provide a framework for high-resolution genetic analysis in oat, and a model for marker development and map construction in other species with complex genomes and limited resources.

Quantitative trait locus mapping of genes under selection across multiple years and sites in Avena barbata: epistasis, pleiotropy, and genotype-by-environment interactions.

The genetic architecture of variation in evolutionary fitness determines the trajectory of adaptive change. We identified quantitative trait loci (QTL) affecting fitness in a mapping population of recombinant inbred lines (RILs) derived from a cross between moist- and dry- associated ecotypes of Avena barbata. We estimated fitness in 179 RILs in each of two natural environments in each of 4 years. Two loci account for over half of the variation in geometric mean fitness across environments. These loci are associated in repulsion phase in the wild ecotypes, suggesting the potential for strong transgressive segregation, but also show significant epistasis giving hybrid breakdown. This epistasis is the result of sharply lower fitness in only one of the recombinant genotypes, suggesting that the loci may contain synergistically acting mutations. Within each trial (year/site combination), we can explain less of the variation than for geometric mean fitness, but the two major loci are associated with variation in fitness in most environments. Tests for pleiotropic effects of QTL on fitness in different environments reveal that the same loci are under selection in all trials. Genotype-by-environment interactions are significant for some loci, but this reflects variation in the strength, not the direction of selection.

Natural selection on pleiotropic quantitative trait Loci affecting a life-history trade-off in Avena barbata.

We applied QTL mapping to fitness variation of Avena barbata under well-watered greenhouse conditions. One hundred eighty recombinant inbred lines were assayed for flowering time, total size, mass allocation, and fitness. Composite Interval Mapping identified two to five loci affecting these traits. These were well supported in more powerful Multiple and Bayesian interval mapping analyses that indicated that additional QTL, as well as epistatic interactions also affect the traits. The posterior distribution of the number of QTL peaked at five to eight additive loci and one to two interactions, but the specific locations of the additional loci could not be determined with certainty. In most cases in which loci for separate traits mapped to similar locations, explicit tests supported pleiotropy over close linkage of separate loci. Alleles that hastened first flowering generally reduced vegetative mass, increased reproductive mass, and were associated with high fitness. Because effects on mass allocation generally cancelled one another, few loci affected total plant size. Only one QTL affected vegetative mass independent of reproductive mass and this locus had little effect on fitness. Thus selection acts to shift the mass allocation toward greater reproductive allocation, because the correlated decrease in vegetative mass poses only a minor fitness cost.

Shared quantitative trait loci underlying the genetic correlation between continuous traits.

We review genetic correlations among quantitative traits in light of their underlying quantitative trait loci (QTL). We derive an expectation of genetic correlation from the effects of underlying loci and test whether published genetic correlations can be explained by the QTL underlying the traits. While genetically correlated traits shared more QTL (33%) on average than uncorrelated traits (11%), the actual number of shared QTL shared was small. QTL usually predicted the sign of the correlation with good accuracy, but the quantitative prediction was poor. Approximately 25% of trait pairs in the data set had at least one QTL with antagonistic effects. Yet a significant minority (20%) of such trait pairs have net positive genetic correlations due to such antagonistic QTL 'hidden' within positive genetic correlations. We review the evidence on whether shared QTL represent single pleiotropic loci or closely linked monotropic genes, and argue that strict pleiotropy can be viewed as one end of a continuum of recombination rates where r=0. QTL studies of genetic correlation will likely be insufficient to predict evolutionary trajectories over long time spans in large panmictic populations, but will provide important insights into the trade-offs involved in population and species divergence.

Hybridization, recombination, and the genetic basis of fitness variation across environments in Avena barbata.

We created Recombinant Inbred Lines (RILs) derived from a cross between ecotypes of Avena barbata associated with moist (mesic) and dry (xeric) habitats in California. Traits which were correlated with fitness across RILs mapped to the same Quantitative Trait Loci (QTLs) as fitness. However, different QTL affected fitness in different environments so that fitness was weakly correlated across environments. Recombination released considerable heritable variation both in fitness, and in ecologically relevant traits. Many traits showed transgressive segregation caused by recombination of QTL associated in repulsion phase in the parents. In addition, some traits were uncorrelated, allowing novel combinations of those traits to be created. Recombination also created heritable variation in reaction norms for at least one trait (root allocation). Altogether these results suggest that recombination can combine the most selectively advantageous genes and traits of the parents to produce broadly adapted genotypes that are capable of outperforming the parents. Indeed, two of the RILs showed higher fitness than the parental ecotypes across a range of environmental treatments in the greenhouse, but their superiority was less pronounced in the field. Although late-generation recombinants exhibited hybrid breakdown, being less fit, on average, than the mid-parent, early generation hybrids appear to exhibit hybrid vigour through the expression of dominance effects in the heterozyotes. This vigour may offset the effects of hybrid breakdown in the early generations following a cross, enhancing the opportunity for recombination to create broadly adapted genotypes. We discuss the implications of these findings to the evolution of colonizing species.

Identifying loci under selection across contrasting environments in Avena barbata using quantitative trait locus mapping.

We constructed recombinant inbred lines of a cross between naturally occurring ecotypes of Avena barbata (Pott ex Link), Poaceae, associated with contrasting moisture environments. These lines were assessed for fitness in common garden reciprocal transplant experiments in two contrasting field sites in each of two years, as well as a novel, benign greenhouse environment. An AFLP (amplified fragment length polymorphism) linkage map of 129 markers spanned 644 cM in 19 linkage groups, which is smaller, with more linkage groups, than expected. Therefore parts of the A. barbata genome remain unmapped, possibly because they lack variation between the ecotypes. Nevertheless, we identified QTL (quantitative trait loci) under selection in both native environments and in the greenhouse. Across years at the same site, the same loci remain under selection, for the same alleles. Across sites, an overlapping set of loci are under selection with either (i) the same alleles favoured at both sites or (ii) loci under selection at one site and neutral at the other. QTL under selection in the greenhouse were generally unlinked to those under selection in the field because selection acted on a different trait. We found little evidence that selection favours alternate alleles in alternate environments, which would be necessary if genotype by environment interaction were to maintain genetic variation in A. barbata. Additive effect QTL were best able to explain the genetic variation among recombinant inbred lines for the greenhouse environment where heritability was highest, and past selection had not eliminated variation.