Genomic regions associated with chocolate spot (Botrytis fabae Sard.) resistance in faba bean (Vicia faba L.).

Chocolate spot (CS), caused by Botrytis fabae Sard., is an important threat to global faba bean production. Growing resistant faba bean cultivars is, therefore, paramount to preventing yield loss. To date, there have been no reported quantitative trait loci (QTL) associated with CS resistance in faba bean. The objective of this study was to identify genomic regions associated with CS resistance using a recombinant inbred line (RIL) population derived from resistant accession ILB 938. A total of 165 RILs from the cross Mélodie/2 × ILB 938/2 were genotyped and evaluated for CS reactions under replicated controlled climate conditions. The RIL population showed significant variation in response to CS resistance. QTL analysis identified five loci contributing to CS resistance on faba bean chromosomes 1 and 6, accounting for 28.4% and 12.5%, respectively, of the total phenotypic variance. The results of this study not only provide insight into disease-resistance QTL, but also can be used as potential targets for marker-assisted breeding in faba bean genetic improvement for CS resistance. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01307-7.

The genome of cowpea (Vigna unguiculata [L.] Walp.).

Cowpea (Vigna unguiculata [L.] Walp.) is a major crop for worldwide food and nutritional security, especially in sub-Saharan Africa, that is resilient to hot and drought-prone environments. An assembly of the single-haplotype inbred genome of cowpea IT97K-499-35 was developed by exploiting the synergies between single-molecule real-time sequencing, optical and genetic mapping, and an assembly reconciliation algorithm. A total of 519 Mb is included in the assembled sequences. Nearly half of the assembled sequence is composed of repetitive elements, which are enriched within recombination-poor pericentromeric regions. A comparative analysis of these elements suggests that genome size differences between Vigna species are mainly attributable to changes in the amount of Gypsy retrotransposons. Conversely, genes are more abundant in more distal, high-recombination regions of the chromosomes; there appears to be more duplication of genes within the NBS-LRR and the SAUR-like auxin superfamilies compared with other warm-season legumes that have been sequenced. A surprising outcome is the identification of an inversion of 4.2 Mb among landraces and cultivars, which includes a gene that has been associated in other plants with interactions with the parasitic weed Striga gesnerioides. The genome sequence facilitated the identification of a putative syntelog for multiple organ gigantism in legumes. A revised numbering system has been adopted for cowpea chromosomes based on synteny with common bean (Phaseolus vulgaris). An estimate of nuclear genome size of 640.6 Mbp based on cytometry is presented.

High-throughput retrotransposon-based genetic diversity of maize germplasm assessment and analysis.

Maize is one of the world's most important crops and a model for grass genome research. Long terminal repeat (LTR) retrotransposons comprise most of the maize genome; their ability to produce new copies makes them efficient high-throughput genetic markers. Inter-retrotransposon-amplified polymorphisms (IRAPs) were used to study the genetic diversity of maize germplasm. Five LTR retrotransposons (Huck, Tekay, Opie, Ji, and Grande) were chosen, based on their large number of copies in the maize genome, whereas polymerase chain reaction primers were designed based on consensus LTR sequences. The LTR primers showed high quality and reproducible DNA fingerprints, with a total of 677 bands including 392 polymorphic bands showing 58% polymorphism between maize hybrid lines. These markers were used to identify genetic similarities among all lines of maize. Analysis of genetic similarity was carried out based on polymorphic amplicon profiles and genetic similarity phylogeny analysis. This diversity was expected to display ecogeographical patterns of variation and local adaptation. The clustering method showed that the varieties were grouped into three clusters differing in ecogeographical origin. Each of these clusters comprised divergent hybrids with convergent characters. The clusters reflected the differences among maize hybrids and were in accordance with their pedigree. The IRAP technique is an efficient high-throughput genetic marker-generating method.

Long Tandem Arrays of Cassandra Retroelements and Their Role in Genome Dynamics in Plants.

Retrotransposable elements are widely distributed and diverse in eukaryotes. Their copy number increases through reverse-transcription-mediated propagation, while they can be lost through recombinational processes, generating genomic rearrangements. We previously identified extensive structurally uniform retrotransposon groups in which no member contains the gag, pol, or env internal domains. Because of the lack of protein-coding capacity, these groups are non-autonomous in replication, even if transcriptionally active. The Cassandra element belongs to the non-autonomous group called terminal-repeat retrotransposons in miniature (TRIM). It carries 5S RNA sequences with conserved RNA polymerase (pol) III promoters and terminators in its long terminal repeats (LTRs). Here, we identified multiple extended tandem arrays of Cassandra retrotransposons within different plant species, including ferns. At least 12 copies of repeated LTRs (as the tandem unit) and internal domain (as a spacer), giving a pattern that resembles the cellular 5S rRNA genes, were identified. A cytogenetic analysis revealed the specific chromosomal pattern of the Cassandra retrotransposon with prominent clustering at and around 5S rDNA loci. The secondary structure of the Cassandra retroelement RNA is predicted to form super-loops, in which the two LTRs are complementary to each other and can initiate local recombination, leading to the tandem arrays of Cassandra elements. The array structures are conserved for Cassandra retroelements of different species. We speculate that recombination events similar to those of 5S rRNA genes may explain the wide variation in Cassandra copy number. Likewise, the organization of 5S rRNA gene sequences is very variable in flowering plants; part of what is taken for 5S gene copy variation may be variation in Cassandra number. The role of the Cassandra 5S sequences remains to be established.

The pseudogenes of barley.

Pseudogenes have a reputation of being 'evolutionary relics' or 'junk DNA'. While they are well characterized in mammals, studies in more complex plant genomes have so far been hampered by the absence of reference genome sequences. Barley is one of the economically most important cereals and has a genome size of 5.1 Gb. With the first high-quality genome reference assembly available for a Triticeae crop, we conducted a whole-genome assessment of pseudogenes on the barley genome. We identified, characterized and classified 89 440 gene fragments and pseudogenes scattered along the chromosomes, with occasional hotspots and higher densities at the chromosome ends. Full-length pseudogenes (11 015) have preferentially retained their exon-intron structure. Retrotransposition of processed mRNAs only plays a marginal role in their creation. However, the distribution of retroposed pseudogenes reflects the Rabl configuration of barley chromosomes and thus hints at founding mechanisms. While parent genes related to the defense-response were found to be under-represented in cultivated barley, we detected several defense-related pseudogenes in wild barley accessions. The percentage of transcriptionally active pseudogenes is 7.2%, and these may potentially adopt new regulatory roles.The barley genome is rich in pseudogenes and small gene fragments mainly located towards chromosome tips or as tandemly repeated units. Our results indicate non-random duplication and pseudogenization preferences and improve our understanding of the dynamics of gene birth and death in large plant genomes and the mechanisms that lead to evolutionary innovations.

Contribution of crop model structure, parameters and climate projections to uncertainty in climate change impact assessments.

Climate change impact assessments are plagued with uncertainties from many sources, such as climate projections or the inadequacies in structure and parameters of the impact model. Previous studies tried to account for the uncertainty from one or two of these. Here, we developed a triple-ensemble probabilistic assessment using seven crop models, multiple sets of model parameters and eight contrasting climate projections together to comprehensively account for uncertainties from these three important sources. We demonstrated the approach in assessing climate change impact on barley growth and yield at Jokioinen, Finland in the Boreal climatic zone and Lleida, Spain in the Mediterranean climatic zone, for the 2050s. We further quantified and compared the contribution of crop model structure, crop model parameters and climate projections to the total variance of ensemble output using Analysis of Variance (ANOVA). Based on the triple-ensemble probabilistic assessment, the median of simulated yield change was -4% and +16%, and the probability of decreasing yield was 63% and 31% in the 2050s, at Jokioinen and Lleida, respectively, relative to 1981-2010. The contribution of crop model structure to the total variance of ensemble output was larger than that from downscaled climate projections and model parameters. The relative contribution of crop model parameters and downscaled climate projections to the total variance of ensemble output varied greatly among the seven crop models and between the two sites. The contribution of downscaled climate projections was on average larger than that of crop model parameters. This information on the uncertainty from different sources can be quite useful for model users to decide where to put the most effort when preparing or choosing models or parameters for impact analyses. We concluded that the triple-ensemble probabilistic approach that accounts for the uncertainties from multiple important sources provide more comprehensive information for quantifying uncertainties in climate change impact assessments as compared to the conventional approaches that are deterministic or only account for the uncertainties from one or two of the uncertainty sources.

Copy-number variation of housekeeping gene rpl13a in rat strains selected for nervous system excitability.

We evaluated copy number variation (CNV) for four genes in rat strains differing in nervous system excitability. rpl13a copy number is significantly reduced in hippocampus and bone marrow in rats with a high excitability threshold and stress. The observed phenomenon may be associated with a role for rpl13a in lipid metabolism.

Development of IRAP- and REMAP-derived SCAR markers for marker-assisted selection of the stripe rust resistance gene Yr15 derived from wild emmer wheat.

KEY MESSAGE: Yr15 provides broad resistance to stripe rust, an important wheat disease. REMAP- and IRAP-derived co-dominant SCAR markers were developed and localize Yr15 to a 1.2 cM interval. They are reliable across many cultivars. Stripe rust [Pucinia striiformis f.sp. tritici (Pst)] is one of the most important fungal diseases of wheat, found on all continents and in over 60 countries. Wild emmer wheat (Triticum dicoccoides), which is the tetraploid progenitor of durum wheat, is a valuable source of novel stripe rust resistance genes for wheat breeding. T. dicoccoides accession G25 carries Yr15 on chromosome 1BS. Yr15 confers resistance to virtually all tested Pst isolates; it is effective in durum and bread wheat introgressions and their derivatives. Retrotransposons generate polymorphic insertions, which can be scored as Mendelian markers using techniques such as REMAP and IRAP. Six REMAP- and IRAP-derived SCAR markers were mapped using 1,256 F2 plants derived from crosses of the susceptible T. durum accession D447 (DW1) with its resistant BC3F9 and BC3F10 (B9 and B10) near isogenic lines, which carried Yr15 introgressed from G25. The nearest markers segregated 0.1 cM proximally and 1.1 cM distally to Yr15. These markers were also mapped and validated at the same position in another 500 independent F2 plants derived from crosses of B9 and B10 with the susceptible cultivar Langdon (LDN). SC2700 and SC790, defining Yr15 on an interval of 1.2 cM, were found to be reliable and robust co-dominant markers in a wide range of wheat lines and cultivars with and without Yr15. These markers are useful tags in marker-assisted wheat breeding programs that aim to incorporate Yr15 into elite wheat lines and cultivars for durable and broad-spectrum resistance to stripe rust.

Anchoring and ordering NGS contig assemblies by population sequencing (POPSEQ).

Next-generation whole-genome shotgun assemblies of complex genomes are highly useful, but fail to link nearby sequence contigs with each other or provide a linear order of contigs along individual chromosomes. Here, we introduce a strategy based on sequencing progeny of a segregating population that allows de novo production of a genetically anchored linear assembly of the gene space of an organism. We demonstrate the power of the approach by reconstructing the chromosomal organization of the gene space of barley, a large, complex and highly repetitive 5.1 Gb genome. We evaluate the robustness of the new assembly by comparison to a recently released physical and genetic framework of the barley genome, and to various genetically ordered sequence-based genotypic datasets. The method is independent of the need for any prior sequence resources, and will enable rapid and cost-efficient establishment of powerful genomic information for many species.

The Tvv1 retrotransposon family is conserved between plant genomes separated by over 100 million years.

KEY MESSAGE: Combining several different approaches, we have examined the structure, variability, and distribution of Tvv1 retrotransposons. Tvv1 is an unusual example of a low-copy retrotransposon metapopulation dispersed unevenly among very distant species and is promising for the development of molecular markers. Retrotransposons are ubiquitous throughout the genomes of the vascular plants, but individual retrotransposon families tend to be confined to the level of plant genus or at most family. This restricts the general applicability of a family as molecular markers. Here, we characterize a new plant retrotransposon named Tvv1_Sdem, a member of the Copia superfamily of LTR retrotransposons, from the genome of the wild potato Solanum demissum. Comparative analyses based on structure and sequence showed a high level of similarity of Tvv1_Sdem with Tvv1-VB, a retrotransposon previously described in the grapevine genome Vitis vinifera. Extending the analysis to other species by in silico and in vitro approaches revealed the presence of Tvv1 family members in potato, tomato, and poplar genomes, and led to the identification of full-length copies of Tvv1 in these species. We were also able to identify polymorphism in UTL sequences between Tvv1_Sdem copies from wild and cultivated potatoes that are useful as molecular markers. Combining different approaches, our results suggest that the Tvv1 family of retrotransposons has a monophyletic origin and has been maintained in both the rosids and the asterids, the major clades of dicotyledonous plants, since their divergence about 100 MYA. To our knowledge, Tvv1 represents an unusual plant retrotransposon metapopulation comprising highly similar members disjointedly dispersed among very distant species. The twin features of Tvv1 presence in evolutionarily distant genomes and the diversity of its UTL region in each species make it useful as a source of robust molecular markers for diversity studies and breeding.

Retrotransposon BARE displays strong tissue-specific differences in expression.

The BARE retrotransposon comprises c. 10% of the barley (Hordeum vulgare) genome. It is actively transcribed, translated and forms virus-like particles (VLPs). For retrotransposons, the inheritance of new copies depends critically on where in the plant replication occurs. In order to shed light on the replication strategy of BARE in the plant, we have used immunolocalization and in situ hybridization to examine expression of the BARE capsid protein, Gag, at a tissue-specific level. Gag is expressed in provascular tissues and highly localized in companion cells surrounding the phloem sieve tubes in mature vascular tissues. BARE Gag and RNA was not seen in the shoot apical meristem of young seedlings, but appeared, following transition to flowering, in the developing floral spike. Moreover, Gag has a highly specific localization in pre-fertilization ovaries. The strong presence of Gag in the floral meristems suggests that newly replicated copies there will be passed to the next generation. BARE expression patterns are consistent with transcriptional regulation by predicted response elements in the BARE promoter, and in the ovary with release from epigenetic transcriptional silencing. To our knowledge, this is the first analysis of the expression of native retrotransposon proteins within a plant to be reported.

A unified classification system for eukaryotic transposable elements.

Our knowledge of the structure and composition of genomes is rapidly progressing in pace with their sequencing. The emerging data show that a significant portion of eukaryotic genomes is composed of transposable elements (TEs). Given the abundance and diversity of TEs and the speed at which large quantities of sequence data are emerging, identification and annotation of TEs presents a significant challenge. Here we propose the first unified hierarchical classification system, designed on the basis of the transposition mechanism, sequence similarities and structural relationships, that can be easily applied by non-experts. The system and nomenclature is kept up to date at the WikiPoson web site.

Drought and recovery in barley: key gene networks and retrotransposon response.

Introduction: During drought, plants close their stomata at a critical soil water content (SWC), together with making diverse physiological, developmental, and biochemical responses. Methods: Using precision-phenotyping lysimeters, we imposed pre-flowering drought on four barley varieties (Arvo, Golden Promise, Hankkija 673, and Morex) and followed their physiological responses. For Golden Promise, we carried out RNA-seq on leaf transcripts before and during drought and during recovery, also examining retrotransposon BARE1expression. Transcriptional data were subjected to network analysis. Results: The varieties differed by their critical SWC (Ï´crit), Hankkija 673 responding at the highest and Golden Promise at the lowest. Pathways connected to drought and salinity response were strongly upregulated during drought; pathways connected to growth and development were strongly downregulated. During recovery, growth and development pathways were upregulated; altogether, 117 networked genes involved in ubiquitin-mediated autophagy were downregulated. Discussion: The differential response to SWC suggests adaptation to distinct rainfall patterns. We identified several strongly differentially expressed genes not earlier associated with drought response in barley. BARE1 transcription is strongly transcriptionally upregulated by drought and downregulated during recovery unequally between the investigated cultivars. The downregulation of networked autophagy genes suggests a role for autophagy in drought response; its importance to resilience should be further investigated.

Drought response of water-conserving and non-conserving spring barley cultivars.

Introduction: Breeding barley cultivars adapted to drought requires in-depth knowledge on physiological drought responses. Methods: We used a high-throughput functional phenotyping platform to examine the response of four high-yielding European spring barley cultivars to a standardized drought treatment imposed around flowering. Results: Cv. Chanell showed a non-conserving water-use behavior with high transpiration and maximum productivity under well-watered conditions but rapid transpiration decrease under drought. The poor recovery upon re-irrigation translated to large yield losses. Cv. Baronesse showed the most water-conserving behavior, with the lowest pre-drought transpiration and the most gradual transpiration reduction under drought. Its good recovery (resilience) prevented large yield losses. Cv. Formula was less conserving than cv. Baronesse and produced low yet stable yields. Cv. RGT's dynamic water use with high transpiration under ample water supply and moderate transpiration decrease under drought combined with high resilience secured the highest and most stable yields. Discussion: Such a dynamic water-use behavior combined with higher drought resilience and favorable root traits could potentially create an ideotype for intermediate drought. Prospective studies will examine these results in field experiments and will use the newly gained understanding on water use in barley to improve process descriptions in crop simulation models to support crop model-aided ideotype design.

Java web tools for PCR, in silico PCR, and oligonucleotide assembly and analysis.

The polymerase chain reaction is fundamental to molecular biology and is the most important practical molecular technique for the research laboratory. We have developed and tested efficient tools for PCR primer and probe design, which also predict oligonucleotide properties based on experimental studies of PCR efficiency. The tools provide comprehensive facilities for designing primers for most PCR applications and their combinations, including standard, multiplex, long-distance, inverse, real-time, unique, group-specific, bisulphite modification assays, Overlap-Extension PCR Multi-Fragment Assembly, as well as a programme to design oligonucleotide sets for long sequence assembly by ligase chain reaction. The in silico PCR primer or probe search includes comprehensive analyses of individual primers and primer pairs. It calculates the melting temperature for standard and degenerate oligonucleotides including LNA and other modifications, provides analyses for a set of primers with prediction of oligonucleotide properties, dimer and G-quadruplex detection, linguistic complexity, and provides a dilution and resuspension calculator.

Multi-environment genome -wide association mapping of culm morphology traits in barley.

In cereals with hollow internodes, lodging resistance is influenced by morphological characteristics such as internode diameter and culm wall thickness. Despite their relevance, knowledge of the genetic control of these traits and their relationship with lodging is lacking in temperate cereals such as barley. To fill this gap, we developed an image analysis-based protocol to accurately phenotype culm diameters and culm wall thickness across 261 barley accessions. Analysis of culm trait data collected from field trials in seven different environments revealed high heritability values (>50%) for most traits except thickness and stiffness, as well as genotype-by-environment interactions. The collection was structured mainly according to row-type, which had a confounding effect on culm traits as evidenced by phenotypic correlations. Within both row-type subsets, outer diameter and section modulus showed significant negative correlations with lodging (<-0.52 and <-0.45, respectively), but no correlation with plant height, indicating the possibility of improving lodging resistance independent of plant height. Using 50k iSelect SNP genotyping data, we conducted multi-environment genome-wide association studies using mixed model approach across the whole panel and row-type subsets: we identified a total of 192 quantitative trait loci (QTLs) for the studied traits, including subpopulation-specific QTLs and 21 main effect loci for culm diameter and/or section modulus showing effects on lodging without impacting plant height. Providing insights into the genetic architecture of culm morphology in barley and the possible role of candidate genes involved in hormone and cell wall-related pathways, this work supports the potential of loci underpinning culm features to improve lodging resistance and increase barley yield stability under changing environments.

Cassandra retrotransposons carry independently transcribed 5S RNA.

We report a group of TRIMs (terminal-repeat retrotransposons in miniature), which are small nonautonomous retrotransposons. These elements, named Cassandra, universally carry conserved 5S RNA sequences and associated RNA polymerase (pol) III promoters and terminators in their long terminal repeats (LTRs). They were found in all vascular plants investigated. Uniquely for LTR retrotransposons, Cassandra produces noncapped, polyadenylated transcripts from the 5S pol III promoter. Capped, read-through transcripts containing Cassandra sequences can also be detected in RNA and in EST databases. The predicted Cassandra RNA 5S secondary structures resemble those for cellular 5S rRNA, with high information content specifically in the pol III promoter region. Genic integration sites are common for Cassandra, an unusual feature for abundant retrotransposons. The 5S in each LTR produces a tandem 5S arrangement with an inter-5S spacing resembling that of cellular 5S. The distribution of 5S genes is very variable in flowering plants and may be partially explained by Cassandra activity. Cassandra thus appears both to have adapted a ubiquitous cellular gene for ribosomal RNA for use as a promoter and to parasitize an as-yet-unidentified group of retrotransposons for the proteins needed in its lifecycle.

Palindromic Sequence-Targeted (PST) PCR, Version 2: An Advanced Method for High-Throughput Targeted Gene Characterization and Transposon Display.

Genome walking (GW), a strategy for capturing previously unsequenced DNA fragments that are in proximity to a known sequence tag, is currently predominantly based on PCR. Recently developed PCR-based methods allow for combining of sequence-specific primers with designed capturing primers capable of annealing to unknown DNA targets, thereby offering the rapidity and effectiveness of PCR. This study presents a methodological improvement to the previously described GW technique known as palindromic sequence-targeted PCR (PST-PCR). Like PST-PCR, this new method (called PST-PCR v.2) relies on targeting of capturing primers to palindromic sequences arbitrarily present in natural DNA templates. PST-PCR v.2 consists of two rounds of PCR. The first round uses a combination of one sequence-specific primer with one capturing (PST) primer. The second round uses a combination of a single (preferred) or two universal primers; one anneals to a 5' tail attached to the sequence-specific primer and the other anneals to a different 5' tail attached to the PST primer. The key advantage of PST-PCR v.2 is the convenience of using a single universal primer with invariable sequences in GW processes involving various templates. The entire procedure takes approximately 2-3 h to produce the amplified PCR fragment, which contains a portion of a template flanked by the sequence-specific and capturing primers. PST-PCR v.2 is highly suitable for simultaneous work with multiple samples. For this reason, PST-PCR v.2 can be applied beyond the classical task of GW for studies in population genetics, in which PST-PCR v.2 is a preferred alternative to amplified fragment length polymorphism (AFLP) or next-generation sequencing. Furthermore, the conditions for PST-PCR v.2 are easier to optimize, as only one sequence-specific primer is used. This reduces non-specific random amplified polymorphic DNA (RAPD)-like amplification and formation of non-templated amplification. Importantly, akin to the previous version, PST-PCR v.2 is not sensitive to template DNA sequence complexity or quality. This study illustrates the utility of PST-PCR v.2 for transposon display (TD), which is a method to characterize inter- or intra-specific variability related to transposon integration sites. The Ac transposon sequence in the maize (Zea mays) genome was used as a sequence tag during the TD procedure to characterize the Ac integration sites.

Transposon-Based Tagging In Silico Using FastPCR Software.

Retrotransposons are ubiquitous, generally dispersed components of eukaryotic genomes. These properties, together with their "copy and paste" lifecycle that generates insertional polymorphism without need for excision, makes them widely useful as a molecular-genetic tags. Various tagging systems have been developed that exploit the sequence conservation of retrotransposon components, such as those found in their long terminal repeats (LTRs). To detect polymorphisms for retrotransposon insertions, marker systems generally rely on PCR amplification between the termini and some component of flanking genomic DNA. As complements to various "wet lab" protocols for retrotransposon tagging, in silico bioinformatics approaches are useful for predicting likely outcomes from unsequenced accessions on the basis of reference genomes. In this chapter, we describe protocols for in silico retrotransposon-based fingerprinting techniques using the FastPCR software as an integrated tools environment for in silico PCR primer design and analysis.