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.

Genetic architecture of inter-specific and -generic grass hybrids by network analysis on multi-omics data.

BACKGROUND: Understanding the mechanisms underlining forage production and its biomass nutritive quality at the omics level is crucial for boosting the output of high-quality dry matter per unit of land. Despite the advent of multiple omics integration for the study of biological systems in major crops, investigations on forage species are still scarce. RESULTS: Our results identified substantial changes in gene co-expression and metabolite-metabolite network topologies as a result of genetic perturbation by hybridizing L. perenne with another species within the genus (L. multiflorum) relative to across genera (F. pratensis). However, conserved hub genes and hub metabolomic features were detected between pedigree classes, some of which were highly heritable and displayed one or more significant edges with agronomic traits in a weighted omics-phenotype network. In spite of tagging relevant biological molecules as, for example, the light-induced rice 1 (LIR1), hub features were not necessarily better explanatory variables for omics-assisted prediction than features stochastically sampled and all available regressors. CONCLUSIONS: The utilization of computational techniques for the reconstruction of co-expression networks facilitates the identification of key omic features that serve as central nodes and demonstrate correlation with the manifestation of observed traits. Our results also indicate a robust association between early multi-omic traits measured in a greenhouse setting and phenotypic traits evaluated under field conditions.

Chromosome-scale assembly and annotation of the perennial ryegrass genome.

BACKGROUND: The availability of chromosome-scale genome assemblies is fundamentally important to advance genetics and breeding in crops, as well as for evolutionary and comparative genomics. The improvement of long-read sequencing technologies and the advent of optical mapping and chromosome conformation capture technologies in the last few years, significantly promoted the development of chromosome-scale genome assemblies of model plants and crop species. In grasses, chromosome-scale genome assemblies recently became available for cultivated and wild species of the Triticeae subfamily. Development of state-of-the-art genomic resources in species of the Poeae subfamily, which includes important crops like fescues and ryegrasses, is lagging behind the progress in the cereal species. RESULTS: Here, we report a new chromosome-scale genome sequence assembly for perennial ryegrass, obtained by combining PacBio long-read sequencing, Illumina short-read polishing, BioNano optical mapping and Hi-C scaffolding. More than 90% of the total genome size of perennial ryegrass (approximately 2.55 Gb) is covered by seven pseudo-chromosomes that show high levels of collinearity to the orthologous chromosomes of Triticeae species. The transposon fraction of perennial ryegrass was found to be relatively low, approximately 35% of the total genome content, which is less than half of the genome repeat content of cultivated cereal species. We predicted 54,629 high-confidence gene models, 10,287 long non-coding RNAs and a total of 8,393 short non-coding RNAs in the perennial ryegrass genome. CONCLUSIONS: The new reference genome sequence and annotation presented here are valuable resources for comparative genomic studies in grasses, as well as for breeding applications and will expedite the development of productive varieties in perennial ryegrass and related species.

Breaking Free: The Genomics of Allopolyploidy-Facilitated Niche Expansion in White Clover.

The merging of distinct genomes, allopolyploidization, is a widespread phenomenon in plants. It generates adaptive potential through increased genetic diversity, but examples demonstrating its exploitation remain scarce. White clover (Trifolium repens) is a ubiquitous temperate allotetraploid forage crop derived from two European diploid progenitors confined to extreme coastal or alpine habitats. We sequenced and assembled the genomes and transcriptomes of this species complex to gain insight into the genesis of white clover and the consequences of allopolyploidization. Based on these data, we estimate that white clover originated ∼15,000 to 28,000 years ago during the last glaciation when alpine and coastal progenitors were likely colocated in glacial refugia. We found evidence of progenitor diversity carryover through multiple hybridization events and show that the progenitor subgenomes have retained integrity and gene expression activity as they traveled within white clover from their original confined habitats to a global presence. At the transcriptional level, we observed remarkably stable subgenome expression ratios across tissues. Among the few genes that show tissue-specific switching between homeologous gene copies, we found flavonoid biosynthesis genes strongly overrepresented, suggesting an adaptive role of some allopolyploidy-associated transcriptional changes. Our results highlight white clover as an example of allopolyploidy-facilitated niche expansion, where two progenitor genomes, adapted and confined to disparate and highly specialized habitats, expanded to a ubiquitous global presence after glaciation-associated allopolyploidization.

Transcriptome profiling and environmental linkage to salinity across Salicornia europaea vegetation.

BACKGROUND: Salicornia europaea, a succulent obligatory halophyte is the most salt-tolerant plant species in the world. It survives salt concentrations of more than 1 M. Therefore, it is a suitable model plant to identify genes involved in salt tolerance mechanisms that can be used for the improvement of crops. The changes in a plant's gene expression in response to abiotic stresses may depend on factors like soil conditions at the site, seasonality, etc. To date, experiments were performed to study the gene expression of S. europaea only under controlled conditions. Conversely, the present study investigates the transcriptome and physicochemical parameters of S. europaea shoots and roots from two different types of saline ecosystems growing under natural conditions. RESULTS: The level of soil salinity was higher at the naturally saline site than at the anthropogenic saline site. The parameters such as ECe, Na+, Cl-, Ca+, SO42- and HCO3- of the soils and plant organs significantly varied according to sites and seasons. We found that Na+ mainly accumulated in shoots, whereas K+ and Ca2+ levels were higher in roots throughout the growing period. Moreover, changes in S. europaea gene expression were more prominent in seasons, than sites and plant organs. The 30 differentially expressed genes included enzymes for synthesis of S-adenosyl methionine, CP47 of light-harvesting complex II, photosystem I proteins, Hsp70 gene, ATP-dependent Clp proteases, ribulose bisphosphate carboxylase/oxygenase (Rubisco), phenylalanine ammonia-lyase (PAL), cytochrome c oxidase (COX) and ATP synthase. CONCLUSION: The comparisons made based on two seasons, plant organs and two different sites suggest the importance of seasonal variations in gene expression of S. europaea. We identify the genes that may play an important role in acclimation to season-dependent changes of salinity. The genes were involved in processes such as osmotic adjustment, energy metabolism and photosynthesis.

Pooled DNA sequencing to identify SNPs associated with a major QTL for bacterial wilt resistance in Italian ryegrass (Lolium multiflorum Lam.).

KEY MESSAGE: SNPs and candidate genes associated with bacterial wilt resistance in Italian ryegrass were identified by sequencing the parental plants and pooled F1 progeny of a segregating population. Italian ryegrass (Lolium multiflorum Lam.) is one of the most important forage grass species in temperate regions. Its yield, quality and persistency can significantly be reduced by bacterial wilt, a serious disease caused by Xanthomonas translucens pv. graminis. Although a major QTL for bacterial wilt resistance has previously been reported, detailed knowledge on underlying genes and DNA markers to allow for efficient resistance breeding strategies is currently not available. We used pooled DNA sequencing to characterize a major QTL for bacterial wilt resistance of Italian ryegrass and to develop inexpensive sequence-based markers to efficiently target resistance alleles for marker-assisted recurrent selection. From the mapping population segregating for the QTL, DNA of 44 of the most resistant and 44 of the most susceptible F1 individuals was pooled and sequenced using the Illumina HiSeq 2000 platform. Allele frequencies of 18 × 106 single nucleotide polymorphisms (SNP) were determined in the resistant and susceptible pool. A total of 271 SNPs on 140 scaffold sequences of the reference parental genome showed significantly different allele frequencies in both pools. We converted 44 selected SNPs to KASP™ markers, genetically mapped these proximal to the major QTL and thus validated their association with bacterial wilt resistance. This study highlights the power of pooled DNA sequencing to efficiently target binary traits in biparental mapping populations. It delivers genome sequence data, SNP markers and potential candidate genes which will allow to implement marker-assisted strategies to fix bacterial wilt resistance in outcrossing breeding populations of Italian ryegrass.

SNP markers associated with body size and pelt length in American mink (Neovison vison).

BACKGROUND: Identification of genes underlying production traits is a key aim of the mink research community. Recent availability of genomic tools have opened the possibility for faster genetic progress in mink breeding. Availability of mink genome assembly allows genome-wide association studies in mink. RESULTS: In this study, we used genotyping-by-sequencing to obtain single nucleotide polymorphism (SNP) genotypes of 2496 mink. After multiple rounds of filtering, we retained 28,336 high quality SNPs and 2352 individuals for a genome-wide association study (GWAS). We performed the first GWAS for body weight, behavior, along with 10 traits related to fur quality in mink. CONCLUSIONS: Combining association results with existing functional information of genes and mammalian phenotype databases, we proposed WWC3, MAP2K4, SLC7A1 and USP22 as candidate genes for body weight and pelt length in mink.

DArT, SNP, and SSR analyses of genetic diversity in Lolium perenne L. using bulk sampling.

BACKGROUND: Lolium perenne L. is the most important forage grass species in temperate regions. It is also considered as a sustainable source of biomass for energy production. However, improvement in biomass yield has been limited by comparison with other major crops. More efficient utilisation of genetic resources and improved breeding schemes are required to advance L. perenne breeding. In an attempt to elucidate the extent of genetic diversity in L. perenne, 1384 DArT, 182 SNP and 48 SSR markers were applied to 297 accessions (Set I) contributed by three German breeding companies and the IPK Genebank. Due to the heterogeneous nature of Lolium accessions, bulk samples were used. Apart from germplasm set I, additional set II and set III was used to determine the reproducibility of marker system and judge the feasibility of bulk strategy in this study. RESULTS: By assessing different bulk sizes, 24 individuals per sample were shown to be a representative number of plants to discriminate different accessions. Among the 297 accessions, all marker types revealed a high polymorphism rate; 1.99, 2.00 and 8.19 alleles, were obtained per locus on average using DArTs, SNPs and SSRs, respectively. The Jaccard distance for DArT markers ranged from 0.00 to 0.73, the Modified Roger's distance (MRD) for SNP markers ranged from 0.03 to 0.52, and for SSR markers from 0.26 to 0.76. Gene diversity for dominant DArT and co-dominant SNP and SSR markers was found to be 0.26, 0.32 and 0.45, respectively. DArT markers showed the highest consistency and reproducibility. CONCLUSION: The resulting data were evaluated using a number of different classification methods, but none of the methods showed a clear differentiation into distinct genetic pools. With regard to hybrid breeding, this will possibly impede substantial progress towards increased biomass yields of L. perenne by utilising heterosis.

A Gene Encoding a DUF247 Domain Protein Cosegregates with the S Self-Incompatibility Locus in Perennial Ryegrass.

The grass family (Poaceae), the fourth largest family of flowering plants, encompasses the most economically important cereal, forage, and energy crops, and exhibits a unique gametophytic self-incompatibility (SI) mechanism that is controlled by at least two multiallelic and independent loci, S and Z. Despite intense research efforts over the last six decades, the genes underlying S and Z remain uncharacterized. Here, we report a fine-mapping approach to identify the male component of the S-locus in perennial ryegrass (Lolium perenne L.) and provide multiple evidence that a domain of unknown function 247 (DUF247) gene is involved in its determination. Using a total of 10,177 individuals from seven different mapping populations segregating for S, we narrowed the S-locus to a genomic region containing eight genes, the closest recombinant marker mapping at a distance of 0.016 cM. Of the eight genes cosegregating with the S-locus, a highly polymorphic gene encoding for a protein containing a DUF247 was fully predictive of known S-locus genotypes at the amino acid level in the seven mapping populations. Strikingly, this gene showed a frameshift mutation in self-compatible darnel (Lolium temulentum L.), whereas all of the self-incompatible species of the Festuca-Lolium complex were predicted to encode functional proteins. Our results represent a major step forward toward understanding the gametophytic SI system in one of the most important plant families and will enable the identification of additional components interacting with the S-locus.

Transcript profiling of a bitter variety of narrow-leafed lupin to discover alkaloid biosynthetic genes.

Lupins (Lupinus spp.) are nitrogen-fixing legumes that accumulate toxic alkaloids in their protein-rich beans. These anti-nutritional compounds belong to the family of quinolizidine alkaloids (QAs), which are of interest to the pharmaceutical and chemical industries. To unleash the potential of lupins as protein crops and as sources of QAs, a thorough understanding of the QA pathway is needed. However, only the first enzyme in the pathway, lysine decarboxylase (LDC), is known. Here, we report the transcriptome of a high-QA variety of narrow-leafed lupin (L. angustifolius), obtained using eight different tissues and two different sequencing technologies. In addition, we present a list of 33 genes that are closely co-expressed with LDC and that represent strong candidates for involvement in lupin alkaloid biosynthesis. One of these genes encodes a copper amine oxidase able to convert the product of LDC, cadaverine, into 1-piperideine, as shown by heterologous expression and enzyme assays. Kinetic analysis revealed a low KM value for cadaverine, supporting a role as the second enzyme in the QA pathway. Our transcriptomic data set represents a crucial step towards the discovery of enzymes, transporters, and regulators involved in lupin alkaloid biosynthesis.

Genomic prediction of starch content and chipping quality in tetraploid potato using genotyping-by-sequencing.

KEY MESSAGE: Genomic prediction models for starch content and chipping quality show promising results, suggesting that genomic selection is a feasible breeding strategy in tetraploid potato. Genomic selection uses genome-wide molecular markers to predict performance of individuals and allows selections in the absence of direct phenotyping. It is regarded as a useful tool to accelerate genetic gain in breeding programs, and is becoming increasingly viable for crops as genotyping costs continue to fall. In this study, we have generated genomic prediction models for starch content and chipping quality in tetraploid potato to facilitate varietal development. Chipping quality was evaluated as the colour of a potato chip after frying following cold induced sweetening. We used genotyping-by-sequencing to genotype 762 offspring, derived from a population generated from biparental crosses of 18 tetraploid parents. Additionally, 74 breeding clones were genotyped, representing a test panel for model validation. We generated genomic prediction models from 171,859 single-nucleotide polymorphisms to calculate genomic estimated breeding values. Cross-validated prediction correlations of 0.56 and 0.73 were obtained within the training population for starch content and chipping quality, respectively, while correlations were lower when predicting performance in the test panel, at 0.30-0.31 and 0.42-0.43, respectively. Predictions in the test panel were slightly improved when including representatives from the test panel in the training population but worsened when preceded by marker selection. Our results suggest that genomic prediction is feasible, however, the extremely high allelic diversity of tetraploid potato necessitates large training populations to efficiently capture the genetic diversity of elite potato germplasm and enable accurate prediction across the entire spectrum of elite potatoes. Nonetheless, our results demonstrate that GS is a promising breeding strategy for tetraploid potato.

A synteny-based draft genome sequence of the forage grass Lolium perenne.

Here we report the draft genome sequence of perennial ryegrass (Lolium perenne), an economically important forage and turf grass species that is widely cultivated in temperate regions worldwide. It is classified along with wheat, barley, oats and Brachypodium distachyon in the Pooideae sub-family of the grass family (Poaceae). Transcriptome data was used to identify 28,455 gene models, and we utilized macro-co-linearity between perennial ryegrass and barley, and synteny within the grass family, to establish a synteny-based linear gene order. The gametophytic self-incompatibility mechanism enables the pistil of a plant to reject self-pollen and therefore promote out-crossing. We have used the sequence assembly to characterize transcriptional changes in the stigma during pollination with both compatible and incompatible pollen. Characterization of the pollen transcriptome identified homologs to pollen allergens from a range of species, many of which were expressed to very high levels in mature pollen grains, and are potentially involved in the self-incompatibility mechanism. The genome sequence provides a valuable resource for future breeding efforts based on genomic prediction, and will accelerate the development of new varieties for more productive grasslands.

Estimating genomic heritabilities at the level of family-pool samples of perennial ryegrass using genotyping-by-sequencing.

KEYMESSAGE: By using the genotyping-by-sequencing method, it is feasible to characterize genomic relationships directly at the level of family pools and to estimate genomic heritabilities from phenotypes scored on family-pools in outbreeding species. Genotyping-by-sequencing (GBS) has recently become a promising approach for characterizing plant genetic diversity on a genome-wide scale. We use GBS to extend the concept of heritability beyond individuals by genotyping family-pool samples by GBS and computing genomic relationship matrices (GRMs) and genomic heritabilities directly at the level of family-pools from pool-frequencies obtained by sequencing. The concept is of interest for species where breeding and phenotyping is not done at the individual level but operates uniquely at the level of (multi-parent) families. As an example we demonstrate the approach using a set of 990 two-parent F2 families of perennial ryegrass (Lolium Perenne). The families were phenotyped as a family-unit in field plots for heading date and crown rust resistance. A total of 728 K single nucleotide polymorphism (SNP) variants were available and were divided in groups of different sequencing depths. GRMs based on GBS data showed diagonal values biased upwards at low sequencing depth, while off-diagonals were little affected by the sequencing depth. Using variants with high sequencing depth, genomic heritability for crown rust resistance was 0.33, and for heading date 0.22, and these genomic heritabilities were biased downwards when using variants with lower sequencing depth. Broad sense heritabilities were 0.61 and 0.66, respectively. Underestimation of genomic heritability at lower sequencing depth was confirmed with simulated data. We conclude that it is feasible to use GBS to describe relationships between family-pools and to estimate genomic heritability directly at the level of F2 family-pool samples, but estimates are biased at low sequencing depth.

Comparative transcriptome analysis within the Lolium/Festuca species complex reveals high sequence conservation.

BACKGROUND: The Lolium-Festuca complex incorporates species from the Lolium genera and the broad leaf fescues, both belonging to the subfamily Pooideae. This subfamily also includes wheat, barley, oat and rye, making it extremely important to world agriculture. Species within the Lolium-Festuca complex show very diverse phenotypes, and many of them are related to agronomically important traits. Analysis of sequenced transcriptomes of these non-model species may shed light on the molecular mechanisms underlying this phenotypic diversity. RESULTS: We have generated de novo transcriptome assemblies for four species from the Lolium-Festuca complex, ranging from 52,166 to 72,133 transcripts per assembly. We have also predicted a set of proteins and validated it with a high-confidence protein database from three closely related species (H. vulgare, B. distachyon and O. sativa). We have obtained gene family clusters for the four species using OrthoMCL and analyzed their inferred phylogenetic relationships. Our results indicate that VRN2 is a candidate gene for differentiating vernalization and non-vernalization types in the Lolium-Festuca complex. Grouping of the gene families based on their BLAST identity enabled us to divide ortholog groups into those that are very conserved and those that are more evolutionarily relaxed. The ratio of the non-synonumous to synonymous substitutions enabled us to pinpoint protein sequences evolving in response to positive selection. These proteins may explain some of the differences between the more stress tolerant Festuca, and the less stress tolerant Lolium species. CONCLUSIONS: Our data presents a comprehensive transcriptome sequence comparison between species from the Lolium-Festuca complex, with the identification of potential candidate genes underlying some important phenotypical differences within the complex (such as VRN2). The orthologous genes between the species have a very high %id (91,61%) and the majority of gene families were shared for all of them. It is likely that the knowledge of the genomes will be largely transferable between species within the complex.

Genomic dissection and prediction of heading date in perennial ryegrass.

BACKGROUND: Genomic selection (GS) has become a commonly used technology in animal breeding. In crops, it is expected to significantly improve the genetic gains per unit of time. So far, its implementation in plant breeding has been mainly investigated in species farmed as homogeneous varieties. Concerning crops farmed in family pools, only a few theoretical studies are currently available. Here, we test the opportunity to implement GS in breeding of perennial ryegrass, using real data from a forage breeding program. Heading date was chosen as a model trait, due to its high heritability and ease of assessment. Genome Wide Association analysis was performed to uncover the genetic architecture of the trait. Then, Genomic Prediction (GP) models were tested and prediction accuracy was compared to the one obtained in traditional Marker Assisted Selection (MAS) methods. RESULTS: Several markers were significantly associated with heading date, some locating within or proximal to genes with a well-established role in floral regulation. GP models gave very high accuracies, which were significantly better than those obtained through traditional MAS. Accuracies were higher when predictions were made from related families and from larger training populations, whereas predicting from unrelated families caused the variance of the estimated breeding values to be biased downwards. CONCLUSIONS: We have demonstrated that there are good perspectives for GS implementation in perennial ryegrass breeding, and that problems resulting from low linkage disequilibrium (LD) can be reduced by the presence of structure and related families in the breeding population. While comprehensive Genome Wide Association analysis is difficult in species with extremely low LD, we did identify variants proximal to genes with a known role in flowering time (e.g. CONSTANS and Phytochrome C).

Changes in Lolium perenne transcriptome during cold acclimation in two genotypes adapted to different climatic conditions.

BACKGROUND: Activation of numerous protective mechanisms during cold acclimation is important for the acquisition of freezing tolerance in perennial ryegrass (Lolium perenne L.). To elucidate the molecular mechanisms of cold acclimation in two genotypes ('Veyo' and 'Falster') of perennial ryegrass from distinct geographical origins, we performed transcriptome profiling during cold acclimation using RNA-Seq. METHODS: We cold-acclimated plants from both genotypes in controlled conditions for a period of 17 days and isolated Total RNA at various time points for high throughput sequencing using Illumina technology. RNA-seq reads were aligned to genotype specific references to identify transcripts with significant changes in expression during cold acclimation. RESULTS: The genes induced were involved in protective mechanisms such as cell response to abiotic stimulus, signal transduction, redox homeostasis, plasma membrane and cell wall modifications, and carbohydrate metabolism in both genotypes. 'Falster' genotype, adapted to cold climates, showed a stronger transcriptional differentiation during cold acclimation, and more differentially expressed transcripts related to stress, signal transduction, response to abiotic stimulus, and metabolic processes compared to 'Veyo'. 'Falster' genotype also showed an induction of more transcripts with sequence homology to fructosyltransferase genes (FTs) and a higher fold induction of fructan in response to low-temperature stress. The circadian rhythm network was perturbed in the 'Veyo' genotype adapted to warmer climates. CONCLUSION: In this study, the differentially expressed genes during cold acclimation, potentially involved in numerous protective mechanisms, were identified in two genotypes of perennial ryegrass from distinct geographical origins. The observation that the circadian rhythm network was perturbed in 'Veyo' during cold acclimation may point to a low adaptability of 'Veyo' to low temperature stresses. This study also revealed the transcriptional mechanisms underlying carbon allocation towards fructan biosynthesis in perennial ryegrass.

The perennial ryegrass GenomeZipper: targeted use of genome resources for comparative grass genomics.

Whole-genome sequences established for model and major crop species constitute a key resource for advanced genomic research. For outbreeding forage and turf grass species like ryegrasses (Lolium spp.), such resources have yet to be developed. Here, we present a model of the perennial ryegrass (Lolium perenne) genome on the basis of conserved synteny to barley (Hordeum vulgare) and the model grass genome Brachypodium (Brachypodium distachyon) as well as rice (Oryza sativa) and sorghum (Sorghum bicolor). A transcriptome-based genetic linkage map of perennial ryegrass served as a scaffold to establish the chromosomal arrangement of syntenic genes from model grass species. This scaffold revealed a high degree of synteny and macrocollinearity and was then utilized to anchor a collection of perennial ryegrass genes in silico to their predicted genome positions. This resulted in the unambiguous assignment of 3,315 out of 8,876 previously unmapped genes to the respective chromosomes. In total, the GenomeZipper incorporates 4,035 conserved grass gene loci, which were used for the first genome-wide sequence divergence analysis between perennial ryegrass, barley, Brachypodium, rice, and sorghum. The perennial ryegrass GenomeZipper is an ordered, information-rich genome scaffold, facilitating map-based cloning and genome assembly in perennial ryegrass and closely related Poaceae species. It also represents a milestone in describing synteny between perennial ryegrass and fully sequenced model grass genomes, thereby increasing our understanding of genome organization and evolution in the most important temperate forage and turf grass species.

Flow sorting and sequencing meadow fescue chromosome 4F.

The analysis of large genomes is hampered by a high proportion of repetitive DNA, which makes the assembly of short sequence reads difficult. This is also the case in meadow fescue (Festuca pratensis), which is known for good abiotic stress resistance and has been used in intergeneric hybridization with ryegrasses (Lolium spp.) to produce Festulolium cultivars. In this work, we describe a new approach to analyze the large genome of meadow fescue, which involves the reduction of sample complexity without compromising information content. This is achieved by dissecting the genome to smaller parts: individual chromosomes and groups of chromosomes. As the first step, we flow sorted chromosome 4F and sequenced it by Illumina with approximately 50× coverage. This provided, to our knowledge, the first insight into the composition of the fescue genome, enabled the construction of the virtual gene order of the chromosome, and facilitated detailed comparative analysis with the sequenced genomes of rice (Oryza sativa), Brachypodium distachyon, sorghum (Sorghum bicolor), and barley (Hordeum vulgare). Using GenomeZipper, we were able to confirm the collinearity of chromosome 4F with barley chromosome 4H and the long arm of chromosome 5H. Several new tandem repeats were identified and physically mapped using fluorescence in situ hybridization. They were found as robust cytogenetic markers for karyotyping of meadow fescue and ryegrass species and their hybrids. The ability to purify chromosome 4F opens the way for more efficient analysis of genomic loci on this chromosome underlying important traits, including freezing tolerance. Our results confirm that next-generation sequencing of flow-sorted chromosomes enables an overview of chromosome structure and evolution at a resolution never achieved before.

Association studies using family pools of outcrossing crops based on allele-frequency estimates from DNA sequencing.

KEY MESSAGE: We propose a method in which GBS data can be conveniently analyzed without calling genotypes. F2 families are frequently used in breeding of outcrossing species, for instance to obtain trait measurements on plots. We propose to perform association studies by obtaining a matching "family genotype" from sequencing a pooled sample of the family, and to directly use allele frequencies computed from sequence read-counts for mapping. We show that, under additivity assumptions, there is a linear relationship between the family phenotype and family allele frequency, and that a regression of family phenotype on family allele frequency will estimate twice the allele substitution effect at a locus. However, medium-to-low sequencing depth causes underestimation of the true allele substitution effect. An expression for this underestimation is derived for the case that parents are diploid, such that F2 families have up to four dosages of every allele. Using simulation studies, estimation of the allele effect from F2-family pools was verified and it was shown that the underestimation of the allele effect is correctly described. The optimal design for an association study when sequencing budget would be fixed is obtained using large sample size and lower sequence depth, and using higher SNP density (resulting in higher LD with causative mutations) and lower sequencing depth. Therefore, association studies using genotyping by sequencing are optimal and use low sequencing depth per sample. The developed framework for association studies using allele frequencies from sequencing can be modified for other types of family pools and is also directly applicable for association studies in polyploids.