Selective sweeps identification in distinct groups of cultivated rye (Secale cereale L.) germplasm provides potential candidate genes for crop improvement.

BACKGROUND: During domestication and subsequent improvement plants were subjected to intensive positive selection for desirable traits. Identification of selection targets is important with respect to the future targeted broadening of diversity in breeding programmes. Rye (Secale cereale L.) is a cereal that is closely related to wheat, and it is an important crop in Central, Eastern and Northern Europe. The aim of the study was (i) to identify diverse groups of rye accessions based on high-density, genome-wide analysis of genetic diversity within a set of 478 rye accessions, covering a full spectrum of diversity within the genus, from wild accessions to inbred lines used in hybrid breeding, and (ii) to identify selective sweeps in the established groups of cultivated rye germplasm and putative candidate genes targeted by selection. RESULTS: Population structure and genetic diversity analyses based on high-quality SNP (DArTseq) markers revealed the presence of three complexes in the Secale genus: S. sylvestre, S. strictum and S. cereale/vavilovii, a relatively narrow diversity of S. sylvestre, very high diversity of S. strictum, and signatures of strong positive selection in S. vavilovii. Within cultivated ryes we detected the presence of genetic clusters and the influence of improvement status on the clustering. Rye landraces represent a reservoir of variation for breeding, and especially a distinct group of landraces from Turkey should be of special interest as a source of untapped variation. Selective sweep detection in cultivated accessions identified 133 outlier positions within 13 sweep regions and 170 putative candidate genes related, among others, to response to various environmental stimuli (such as pathogens, drought, cold), plant fertility and reproduction (pollen sperm cell differentiation, pollen maturation, pollen tube growth), and plant growth and biomass production. CONCLUSIONS: Our study provides valuable information for efficient management of rye germplasm collections, which can help to ensure proper safeguarding of their genetic potential and provides numerous novel candidate genes targeted by selection in cultivated rye for further functional characterisation and allelic diversity studies.

Genetic mapping of the powdery mildew resistance gene Pm7 on oat chromosome 5D.

KEY MESSAGE: Three independent experiments with different genetic backgrounds mapped the resistance gene Pm7 in the oat genome to the distal part of the long arm of chromosome 5D. Resistance of oat to Blumeria graminis DC. f. sp. avenae is an important breeding goal in Central and Western Europe. In this study, the position of the effective and widely used resistance gene Pm7 in the oat genome was determined based on three independent experiments with different genetic backgrounds: genome-wide association mapping in a diverse set of inbred oat lines and binary phenotype mapping in two bi-parental populations. Powdery mildew resistance was assessed in the field as well as by detached leaf tests in the laboratory. Genotyping-by-sequencing was conducted to establish comprehensive genetic fingerprints for subsequent genetic mapping experiments. All three mapping approaches located the gene to the distal part of the long arm of chromosome 5D in the hexaploid oat genome sequences of OT3098 and 'Sang.' Markers from this region were homologous to a region of chromosome 2Ce of the C-genome species, Avena eriantha, the donor of Pm7, which appears to be the ancestral source of a translocated region on the hexaploid chromosome 5D.

Improving Yield and Yield Stability in Winter Rye by Hybrid Breeding.

Rye is the only cross-pollinating small-grain cereal. The unique reproduction biology results in an exceptional complexity concerning genetic improvement of rye by breeding. Rye is a close relative of wheat and has a strong adaptation potential that refers to its mating system, making this overlooked cereal readily adjustable to a changing environment. Rye breeding addresses the emerging challenges of food security associated with climate change. The systematic identification, management, and use of its valuable natural diversity became a feasible option in outbreeding rye only following the establishment of hybrid breeding late in the 20th century. In this article, we review the most recent technological advances to improve yield and yield stability in winter rye. Based on recently released reference genome sequences, SMART breeding approaches are described to counterbalance undesired linkage drag effects of major restorer genes on grain yield. We present the development of gibberellin-sensitive semidwarf hybrids as a novel plant breeding innovation based on an approach that is different from current methods of increasing productivity in rye and wheat. Breeding of new rye cultivars with improved performance and resilience is indispensable for a renaissance of this healthy minor cereal as a homogeneous commodity with cultural relevance in Europe that allows for comparatively smooth but substantial complementation of wheat with rye-based diets, supporting the necessary restoration of the balance between human action and nature.

Association mapping of autumn-seeded rye (Secale cereale L.) reveals genetic linkages between genes controlling winter hardiness and plant development.

Winter field survival (WFS) in autumn-seeded winter cereals is a complex trait associated with low temperature tolerance (LTT), prostrate growth habit (PGH), and final leaf number (FLN). WFS and the three sub-traits were analyzed by a genome-wide association study of 96 rye (Secale cereal L.) genotypes of different origins and winter-hardiness levels. A total of 10,244 single nucleotide polymorphism (SNP) markers were identified by genotyping by sequencing and 259 marker-trait-associations (MTAs; p < 0.01) were revealed by association mapping. The ten most significant SNPs (p < 1.49e-04) associated with WFS corresponded to nine strong candidate genes: Inducer of CBF Expression 1 (ICE1), Cold-regulated 413-Plasma Membrane Protein 1 (COR413-PM1), Ice Recrystallization Inhibition Protein 1 (IRIP1), Jasmonate-resistant 1 (JAR1), BIPP2C1-like protein phosphatase, Chloroplast Unusual Positioning Protein-1 (CHUP1), FRIGIDA-like 4 (FRL4-like) protein, Chalcone Synthase 2 (CHS2), and Phenylalanine Ammonia-lyase 8 (PAL8). Seven of the candidate genes were also significant for one or several of the sub-traits supporting the hypothesis that WFS, LTT, FLN, and PGH are genetically interlinked. The winter-hardy rye genotypes generally carried additional allele variants for the strong candidate genes, which suggested allele diversity was a major contributor to cold acclimation efficiency and consistent high WFS under varying field conditions.

A Genome-Wide Association Study Pinpoints Quantitative Trait Genes for Plant Height, Heading Date, Grain Quality, and Yield in Rye (Secale cereale L.).

Rye is the only cross-pollinating Triticeae crop species. Knowledge of rye genes controlling complex-inherited traits is scarce, which, currently, largely disables the genomics assisted introgression of untapped genetic variation from self-incompatible germplasm collections in elite inbred lines for hybrid breeding. We report on the first genome-wide association study (GWAS) in rye based on the phenotypic evaluation of 526 experimental hybrids for plant height, heading date, grain quality, and yield in 2 years and up to 19 environments. We established a cross-validated NIRS calibration model as a fast, effective, and robust analytical method to determine grain quality parameters. We observed phenotypic plasticity in plant height and tiller number as a resource use strategy of rye under drought and identified increased grain arabinoxylan content as a striking phenotype in osmotically stressed rye. We used DArTseq™ as a genotyping-by-sequencing technology to reduce the complexity of the rye genome. We established a novel high-density genetic linkage map that describes the position of almost 19k markers and that allowed us to estimate a low genome-wide LD based on the assessed genetic diversity in elite germplasm. We analyzed the relationship between plant height, heading date, agronomic, as well as grain quality traits, and genotype based on 20k novel single-nucleotide polymorphism markers. In addition, we integrated the DArTseq™ markers in the recently established 'Lo7' reference genome assembly. We identified cross-validated SNPs in 'Lo7' protein-coding genes associated with all traits studied. These include associations of the WUSCHEL-related homeobox transcription factor DWT1 and grain yield, the DELLA protein gene SLR1 and heading date, the Ethylene overproducer 1-like protein gene ETOL1 and thousand-grain weight, protein and starch content, as well as the Lectin receptor kinase SIT2 and plant height. A Leucine-rich repeat receptor protein kinase and a Xyloglucan alpha-1,6-xylosyltransferase count among the cross-validated genes associated with water-extractable arabinoxylan content. This study demonstrates the power of GWAS, hybrid breeding, and the reference genome sequence in rye genetics research to dissect and identify the function of genes shaping genetic diversity in agronomic and grain quality traits of rye. The described links between genetic causes and phenotypic variation will accelerate genomics-enabled rye improvement.

Chromosome-scale genome assembly provides insights into rye biology, evolution and agronomic potential.

Rye (Secale cereale L.) is an exceptionally climate-resilient cereal crop, used extensively to produce improved wheat varieties via introgressive hybridization and possessing the entire repertoire of genes necessary to enable hybrid breeding. Rye is allogamous and only recently domesticated, thus giving cultivated ryes access to a diverse and exploitable wild gene pool. To further enhance the agronomic potential of rye, we produced a chromosome-scale annotated assembly of the 7.9-gigabase rye genome and extensively validated its quality by using a suite of molecular genetic resources. We demonstrate applications of this resource with a broad range of investigations. We present findings on cultivated rye's incomplete genetic isolation from wild relatives, mechanisms of genome structural evolution, pathogen resistance, low-temperature tolerance, fertility control systems for hybrid breeding and the yield benefits of rye-wheat introgressions.

Detecting Large Chromosomal Modifications Using Short Read Data From Genotyping-by-Sequencing.

Markers linked to agronomic traits are of the prerequisite for molecular breeding. Genotyping-by-sequencing (GBS) data enables to detect small polymorphisms including single nucleotide polymorphisms (SNPs) and short insertions or deletions (InDels) that can be used, for instance, for marker-assisted selection, population genetics, and genome-wide association studies (GWAS). Here, we aim at detecting large chromosomal modifications in barley and wheat based on GBS data. These modifications could be duplications, deletions, substitutions including introgressions as well as alterations of DNA methylation. We demonstrate that GBS coverage analysis is capable to detect Hordeum vulgare/Hordeum bulbosum introgression lines. Furthermore, we identify large chromosomal modifications in barley and wheat collections. Hence, large chromosomal modifications, including introgressions and copy number variations (CNV), can be detected easily and can be used as markers in research and breeding without additional wet-lab experiments.

Gene Expression Profiling and Fine Mapping Identifies a Gibberellin 2-Oxidase Gene Co-segregating With the Dominant Dwarfing Gene Ddw1 in Rye (Secale cereale L.).

The gibberellin (GA)-sensitive dwarfing gene Ddw1 provides an opportunity to genetically reduce plant height in rye. Genetic analysis in a population of recombinant inbred lines confirmed a monogenetic dominant inheritance of Ddw1. Significant phenotypic differences in PH between homo- and heterozygotic genotypes indicate an incomplete dominance of Ddw1. De novo transcriptome sequencing of Ddw1 mutant as well as tall genotypes resulted in 113,547 contigs with an average length of 318 bp covering 36.18 Mbp rye DNA. A hierarchical cluster analysis based on individual groups of rye homologs of functionally characterized rice genes controlling morphological or physiological traits including plant height, flowering time, and source activity identified the gene expression profile of stems at the begin of heading to most comprehensively mirror effects of Ddw1. Genome-wide expression profiling identified 186 transcripts differentially expressed between semi-dwarf and tall genotypes in stems. In total, 29 novel markers have been established and mapped to a 27.2 cM segment in the distal part of the long arm of chromosome 5R. Ddw1 could be mapped within a 0.4 cM interval co-segregating with a marker representing the C20-GA2-oxidase gene ScGA2ox12, that is up-regulated in stems of Ddw1 genotypes. The increased expression of ScGA2ox12 observed in semi-dwarf rye as well as structural alterations in transcript sequences associated with the ScGA2ox12 gene implicate, that Ddw1 is a dominant gain-of-function mutant. Integration of the target interval in the wheat reference genome sequence indicated perfect micro-colinearity between the Ddw1 locus and a 831 kb segment on chromosome 5A, which resides inside of a 11.21 Mb interval carrying the GA-sensitive dwarfing gene Rht12 in wheat. The potential of Ddw1 as a breeder's option to improve lodging tolerance in rye is discussed.

Two Rye Genes Responsible for Abnormal Development of Wheat⁻Rye Hybrids Are Linked in the Vicinity of an Evolutionary Translocation on Chromosome 6R.

The post-zygotic reproductive isolation (RI) in plants is frequently based on the negative interaction of the parental genes involved in plant development. Of special interest is the study of such types of interactions in crop plants, because of the importance of distant hybridization in plant breeding. This study is devoted to map rye genes that are incompatible with wheat, determining the development of the shoot apical meristem in wheat⁻rye hybrids. Linkage analysis of microsatellite loci, as well as genes of embryo lethality (Eml-R1) and hybrid dwarfness (Hdw-R1) was carried out in hybrids of Chinese Spring wheat with recombinant inbred lines as well as interline rye hybrids. Eml-R1 and Hdw-R1 could be mapped proximal and distal of two closely linked EST-SSR markers, Xgrm902 and Xgrm959, on rye chromosome 6R. Both rye genes are located on a segment of chromosome 6R that contains a breakpoint of evolutionary translocation between the ancestral chromosomes of homeologous groups 6 and 3. The obtained results are discussed in relation to genes interacting in developmental pathways as a class of causal genes of RI.

QTL mapping and comparative genome analysis of agronomic traits including grain yield in winter rye.

KEY MESSAGE: Genetic diversity in elite rye germplasm as well as F 2:3 testcross design enables fast QTL mapping to approach genes controlling grain yield, grain weight, tiller number and heading date in rye hybrids. Winter rye (Secale cereale L.) is a multipurpose cereal crop closely related to wheat, which offers the opportunity for a sustainable production of food and feed and which continues to emerge as a renewable energy source for the production of bioethanol and biomethane. Rye contributes to increase agricultural crop species diversity particularly in Central and Eastern Europe. In contrast to other small grain cereals, knowledge on the genetic architecture of complex inherited, agronomic important traits is yet limited for the outbreeding rye. We have performed a QTL analysis based on a F2:3 design and testcross performance of 258 experimental hybrids in multi-environmental field trials. A genetic linkage map covering 964.9 cM based on SSR, conserved-orthologous set (COS), and mixed-phase dominant DArT markers allowed to describe 22 QTL with significant effects for grain yield, heading date, tiller number, and thousand grain weight across seven environments. Using rye COS markers, orthologous segments for these traits have been identified in the rice genome, which carry cloned and functionally characterized rice genes. The initial genome scan described here together with the existing knowledge on candidate genes provides the basis for subsequent analyses of the genetic and molecular mechanisms underlying agronomic important traits in rye.

Fine mapping of the restorer gene Rfp3 from an Iranian primitive rye (Secale cereale L.).

KEY MESSAGE: A comparative genetics approach allowed to precisely determine the map position of the restorer gene Rfp3 in rye and revealed that Rfp3 and the restorer gene Rfm1 in barley reside at different positions in a syntenic 4RL/6HS segment. Cytoplasmic male sterility (CMS) is a reliable and striking genetic mechanism for hybrid seed production. Breeding of CMS-based hybrids in cereals requires the use of effective restorer genes as an indispensable pre-requisite. We report on the fine mapping of a restorer gene for the Pampa cytoplasm in winter rye that has been tapped from the Iranian primitive rye population Altevogt 14160. For this purpose, we have mapped 41 gene-derived markers to a 38.8 cM segment in the distal part of the long arm of chromosome 4R, which carries the restorer gene. Male fertility restoration was comprehensively analyzed in progenies of crosses between a male-sterile tester genotype and 21 recombinant as well as six non-recombinant BC4S2 lines. This approach allowed us to validate the position of this restorer gene, which we have designated Rfp3, on chromosome 4RL. Rfp3 was mapped within a 2.5 cM interval and cosegregated with the EST-derived marker c28385. The gene-derived conserved ortholog set (COS) markers enabled us to investigate the orthology of restorer genes originating from different genetic resources of rye as well as barley. The observed localization of Rfp3 and Rfm1 in a syntenic 4RL/6HS segment asks for further efforts towards cloning of both restorer genes as an option to study the mechanisms of male sterility and fertility restoration in cereals.

Towards a whole-genome sequence for rye (Secale cereale L.).

We report on a whole-genome draft sequence of rye (Secale cereale L.). Rye is a diploid Triticeae species closely related to wheat and barley, and an important crop for food and feed in Central and Eastern Europe. Through whole-genome shotgun sequencing of the 7.9-Gbp genome of the winter rye inbred line Lo7 we obtained a de novo assembly represented by 1.29 million scaffolds covering a total length of 2.8 Gbp. Our reference sequence represents nearly the entire low-copy portion of the rye genome. This genome assembly was used to predict 27 784 rye gene models based on homology to sequenced grass genomes. Through resequencing of 10 rye inbred lines and one accession of the wild relative S. vavilovii, we discovered more than 90 million single nucleotide variants and short insertions/deletions in the rye genome. From these variants, we developed the high-density Rye600k genotyping array with 600 843 markers, which enabled anchoring the sequence contigs along a high-density genetic map and establishing a synteny-based virtual gene order. Genotyping data were used to characterize the diversity of rye breeding pools and genetic resources, and to obtain a genome-wide map of selection signals differentiating the divergent gene pools. This rye whole-genome sequence closes a gap in Triticeae genome research, and will be highly valuable for comparative genomics, functional studies and genome-based breeding in rye.

Effect of a rye dwarfing gene on plant height, heading stage, and Fusarium head blight in triticale (×Triticosecale Wittmack).

KEY MESSAGE: The rye-derived dwarfing gene Ddw1 on chromosome 5R acts in triticale in considerably reducing plant height, increasing FHB severity and delaying heading stage. Triticale, an amphiploid hybrid between durum wheat and rye, is an European cereal mainly grown in Germany, France, Poland, and Belarus for feeding purposes. Dwarfing genes might further improve the genetic potential of triticale concerning lodging resistance and yield. However, they might have pleiotropic effects on other, agronomically important traits including Fusarium head blight. Therefore, we analyzed a population of 199 doubled haploid (DH) lines of the cross HeTi117-06 × Pigmej for plant height, heading stage, and FHB severity across 2 locations and 2 years. The most prominent QTL was detected on chromosome 5R explaining 48, 77, and 71 % of genotypic variation for FHB severity, plant height, and heading stage, respectively. The frequency of recovery in cross validation was ≥90 % for all three traits. Because the markers that detect dwarfing gene Ddw1 in rye are also in our population the most closely linked markers, we assume that this major QTL resembles Ddw1. For FHB severity two, for plant height three, and for heading stage five additional QTL were detected. Caused by the considerable genetic variation for heading stage and FHB severity within the progeny with the dwarfing allele, short-strawed, early heading and FHB-resistant lines can be developed when population size is large enough.

Reticulate evolution of the rye genome.

Rye (Secale cereale) is closely related to wheat (Triticum aestivum) and barley (Hordeum vulgare). Due to its large genome (~8 Gb) and its regional importance, genome analysis of rye has lagged behind other cereals. Here, we established a virtual linear gene order model (genome zipper) comprising 22,426 or 72% of the detected set of 31,008 rye genes. This was achieved by high-throughput transcript mapping, chromosome survey sequencing, and integration of conserved synteny information of three sequenced model grass genomes (Brachypodium distachyon, rice [Oryza sativa], and sorghum [Sorghum bicolor]). This enabled a genome-wide high-density comparative analysis of rye/barley/model grass genome synteny. Seventeen conserved syntenic linkage blocks making up the rye and barley genomes were defined in comparison to model grass genomes. Six major translocations shaped the modern rye genome in comparison to a putative Triticeae ancestral genome. Strikingly dissimilar conserved syntenic gene content, gene sequence diversity signatures, and phylogenetic networks were found for individual rye syntenic blocks. This indicates that introgressive hybridizations (diploid or polyploidy hybrid speciation) and/or a series of whole-genome or chromosome duplications played a role in rye speciation and genome evolution.