Fragments of the key flowering gene <em>GIGANTEA</em> are associated with helitron-type sequences in the Pooideae grass <em>Lolium perenne</em>

BACKGROUND: Helitrons are a class of transposable elements which have been identified in a number of species of plants, animals and fungi. They are unique in their proposed rolling-circle mode of replication, have a highly variable copy-number and have been implicated in the restructuring of coding sequences both by their insertion into existing genes and by their incorporation of transcriptionally competent gene fragments. Helitron discovery depends on identifying associated DNA signature sequences and comprehensive evaluation of helitron contribution to a particular genome requires detailed computational analysis of whole genome sequence. Therefore, the role which helitrons have played in modelling non-model plant genomes is largely unknown. RESULTS: Cloning of the flowering gene GIGANTEA (GI) from a BAC library of the Pooideae grass Lolium perenne (perennial ryegrass) identified the target gene and several GI pseudogene fragments spanning the first five exons. Analysis of genomic sequence 5' and 3' of one these GI fragments revealed motifs consistent with helitron-type transposon insertion, specifically a putative 5'-A↓T-3' insertion site containing 5'-TC and CTAG-3' borders with a sub-terminal 16 bp hairpin. Screening of a BAC library of the closely related grass species Festuca pratensis (meadow fescue) indicated similar helitron-associated GI fragments present in this genome, as well as non-helitron associated GI fragments derived from the same region of GI. In order to investigate the possible extent of ancestral helitron-activity in L. perenne, a methylation-filtered GeneThresher(® )genomic library developed from this species was screened for potential helitron 3' hairpin sequences associated with a 3'-CTRR motif. This identified 7 potential helitron hairpin-types present between at least 9 and 51 times within the L. perenne methylation-filtered library. CONCLUSION: This represents evidence for a possible ancestral role for helitrons in modelling the genomes of Lolium and related species

An investigation of genotype-phenotype association in a festulolium forage grass population containing genome-spanning <i>Festuca pratensis</i> chromosome segments in a <i>Lolium perenne</i> background

Alien chromosome introgression is used for the transfer of beneficial traits in plant breeding. For temperate forage grasses, much of the work in this context has focused on species within the ryegrasses (Lolium spp.) and the closely related fescues (Festuca spp.) particularly with a view to combining high forage quality with reliability and enhanced environmental services. We have analysed a L. perenne (perennial ryegrass) population containing the majority of a F. pratensis (meadow fescue) genome as introgressed chromosome segments to identify a) marker-trait associations for nutrient use and abiotic stress response across the family, and b) to assess the effects of introgression of F. pratensis genomic regions on phenotype. Using container-based assays and a system of flowing solution culture, we looked at phenotype responses, including root growth, to nitrogen and phosphorus status in the growing medium and abiotic stresses within this festulolium family. A number of significant marker/trait associations were identified across the family for root biomass on chromosomes 2, 3 and 5 and for heading date on chromosome 2. Of particular interest was a region on chromosome 2 associated with increased root biomass in phosphorus-limited conditions derived from one of the L. perenne parents. A genotype containing F. pratensis chromosome 4 as a monosomic introgression showed increased tiller number, shoot and root growth and genotypes with F. pratensis chromosome segment introgressions at different ends of chromosome 4 exhibited differential phenotypes across a variety of test conditions. There was also a general negative correlation between the extent of the F. pratensis genome that had been introgressed and root-related trait performances. We conclude that 1) the identification of alleles affecting root growth has potential application in forage grass breeding and, 2) F. pratensis introgressions can enhance quantitative traits, however, introgression can also have more general negative effects

B chromosomes are associated with redistribution of genetic recombination towards lower-recombination chromosomal regions in perennial ryegrass

Supernumerary ‘B’ chromosomes are non-essential components of the genome present in a range of plant and animal species – including many grasses. Within diploid and polyploid ryegrass and fescue species, including the forage grass perennial ryegrass (Lolium perenne L.), the presence of B chromosomes has been reported as influencing both chromosome pairing and chiasma frequencies. In this study, the effects of the presence/absence of B chromosomes on genetic recombination has been investigated through generating DArT marker genetic maps for 6 perennial ryegrass diploid populations, the pollen parents of which contained either two B or zero B chromosomes. Through genetic and cytological analyses of these progenies and their parents we have identified that, while overall cytological estimates of chiasma frequencies were significantly lower in pollen mother cells with two B chromosomes as compared to zero B chromosomes, the recombination frequencies within some marker intervals were actually increased, particularly for marker intervals in lower recombination regions of chromosomes, i.e., pericentromeric regions. Thus, in perennial ryegrass, the presence of two B chromosomes redistributed patterns of meiotic recombination in pollen mother cells in ways which could increase the range of allelic variation available to plant breeders

Integration of gene-based markers in a pearl millet genetic map for identification of candidate genes underlying drought tolerance QTLs

BACKGROUND: Identification of genes underlying drought tolerance (DT) quantitative trait loci (QTLs) will facilitate understanding of molecular mechanisms of drought tolerance, and also will accelerate genetic improvement of pearl millet through marker-assisted selection. We report a map based on genes with assigned functional roles in plant adaptation to drought and other abiotic stresses and demonstrate its use in identifying candidate genes underlying a major DT-QTL. RESULTS: Seventy five single nucleotide polymorphism (SNP) and conserved intron spanning primer (CISP) markers were developed from available expressed sequence tags (ESTs) using four genotypes, H 77/833-2, PRLT 2/89-33, ICMR 01029 and ICMR 01004, representing parents of two mapping populations. A total of 228 SNPs were obtained from 30.5 kb sequenced region resulting in a SNP frequency of 1/134 bp. The positions of major pearl millet linkage group (LG) 2 DT-QTLs (reported from crosses H 77/833-2 × PRLT 2/89-33 and 841B × 863B) were added to the present consensus function map which identified 18 genes, coding for PSI reaction center subunit III, PHYC, actin, alanine glyoxylate aminotransferase, uridylate kinase, acyl-CoA oxidase, dipeptidyl peptidase IV, MADS-box, serine/threonine protein kinase, ubiquitin conjugating enzyme, zinc finger C- × 8-C × 5-C × 3-H type, Hd3, acetyl CoA carboxylase, chlorophyll a/b binding protein, photolyase, protein phosphatase1 regulatory subunit SDS22 and two hypothetical proteins, co-mapping in this DT-QTL interval. Many of these candidate genes were found to have significant association with QTLs of grain yield, flowering time and leaf rolling under drought stress conditions. CONCLUSIONS: We have exploited available pearl millet EST sequences to generate a mapped resource of seventy five new gene-based markers for pearl millet and demonstrated its use in identifying candidate genes underlying a major DT-QTL in this species. The reported gene-based markers represent an important resource for identification of candidate genes for other mapped abiotic stress QTLs in pearl millet. They also provide a resource for initiating association studies using candidate genes and also for comparing the structure and function of distantly related plant genomes such as other Poaceae members

Transmission Frequencies of Introgressed Festuca pratensis Chromosomes and Chromosome Segments in Lolium perenne

The introgression of genetic variation from related species into crops provides an important route by which superior plant varieties can be produced. The primary aim of introgression involves the transfer of a small chromosome segment from a related species into a chromosome of a crop species (via recombination at meiosis) to generate an interspecific recombinant chromosome. Very little is known about the selective pressures that act on the products of interspecific recombination. Seven monosomic substitution lines were developed between Lolium perenne and Festuca pratensis. When each line was backcrossed to L. perenne recombination occurred between the F. pratensis chromosome and its L. perenne homoeologue, resulting in backcross populations carrying L. perenne/F. pratensis recombinant chromosomes. This paper describes the relationship between the frequency of generation of interspecific recombinant chromosomes with the frequency of their transmission to the next generation. The results reveal the presence of neutral, negative, and positive selection pressures for the transmission of F. pratensis chromosomes and L. perenne/F. pratensis recombinant chromosomes through the gametes to the next generation. The type of selection pressure observed depended on which linkage group the F. pratensis chromosome under study was derived from. The implications of these results are discussed

A comparison of shared patterns of differential gene expression and gene ontologies in response to water-stress in roots and leaves of four diverse genotypes of Lolium and Festuca spp. temperate pasture grasses

Ryegrasses (Lolium spp.) and fescues (Festuca spp.) are closely related and widely cultivated perennial forage grasses. As such, resilience in the face of abiotic stresses is an important component of their traits. We have compared patterns of differentially expressed genes (DEGs) in roots and leaves of two perennial ryegrass genotypes and a single genotype of each of a festulolium (predominantly Italian ryegrass) and meadow fescue with the onset of water stress, focussing on overall patterns of DEGs and gene ontology terms (GOs) shared by all four genotypes. Plants were established in a growing medium of vermiculite watered with nutrient solution. Leaf and root material were sampled at 35% (saturation) and, as the medium dried, at 15%, 5% and 1% estimated water contents (EWCs) and RNA extracted. Differential gene expression was evaluated comparing the EWC sampling points from RNAseq data using a combination of analysis methods. For all genotypes, the greatest numbers of DEGs were identified in the 35/1 and 5/1 comparisons in both leaves and roots. In total, 566 leaf and 643 root DEGs were common to all 4 genotypes, though a third of these leaf DEGs were not regulated in the same up/down direction in all 4 genotypes. For roots, the equivalent figure was 1% of the DEGs. GO terms shared by all four genotypes were often enriched by both up- and down-regulated DEGs in the leaf, whereas generally, only by either up- or down-regulated DEGs in the root. Overall, up-regulated leaf DEGs tended to be more genotype-specific than down-regulated leaf DEGs or root DEGs and were also associated with fewer GOs. On average, only 5-15% of the DEGs enriching common GO terms were shared by all 4 genotypes, suggesting considerable variation in DEGs between related genotypes in enacting similar biological processes

A new genome allows the identification of genes associated with natural variation in aluminium tolerance in Brachiaria grasses

Toxic concentrations of aluminium cations and low phosphorus availability are the main yield-limiting factors in acidic soils, which represent half of the potentially available arable land. Brachiaria grasses, which are commonly sown as forage in the tropics because of their resilience and low demand for nutrients, show greater tolerance to high concentrations of aluminium cations (Al3+) than most other grass crops. In this work, we explored the natural variation in tolerance to Al3+ between high and low tolerant Brachiaria species and characterized their transcriptional differences during stress. We identified three QTLs (quantitative trait loci) associated with root vigour during Al3+ stress in their hybrid progeny. By integrating these results with a new Brachiaria reference genome, we identified 30 genes putatively responsible for Al3+ tolerance in Brachiaria. We observed differential expression during stress of genes involved in RNA translation, response signalling, cell wall composition, and vesicle location homologous to aluminiuminduced proteins involved in limiting uptake or localizing the toxin. However, there was limited regulation of malate transporters in Brachiaria, which suggests that exudation of organic acids and other external tolerance mechanisms, common in other grasses, might not be relevant in Brachiaria. The contrasting regulation of RNA translation and response signalling suggests that response timing is critical in high Al3+-tolerant Brachiaria

A Comparison of Differential Gene Expression in Response to the Onset of Water Stress Between Three Hybrid Brachiaria Genotypes

Brachiaria (Trin.) Griseb. (syn. Urochloa P. Beauv.) is a C4 grass genus belonging to the Panicoideae. Native to Africa, these grasses are now widely grown as forages in tropical areas worldwide and are the subject of intensive breeding, particularly in South America. Tolerance to abiotic stresses such as aluminum and drought are major breeding objectives. In this study, we present the transcriptomic profiling of leaves and roots of three Brachiaria interspecific hybrid genotypes with the onset of water stress, Br12/3659-17 (gt-17), Br12/2360-9 (gt-9), and Br12/3868-18 (gt-18), previously characterized as having good, intermediate and poor tolerance to drought, respectively, in germplasm evaluation programs. RNA was extracted from leaf and root tissue of plants at estimated growing medium water contents (EWC) of 35, 15, and 5%. Differentially expressed genes (DEGs) were compared between different EWCs, 35/15, 15/5, and 35/5 using DESeq2. Overall, the proportions of DEGs enriched in all three genotypes varied in a genotype-dependent manner in relation to EWC comparison, with intermediate and sensitive gt-9 and gt-18 being more similar to each other than to drought tolerant gt-17. More specifically, GO terms relating to carbohydrate and cell wall metabolism in the leaves were enriched by up-regulated DEGs in gt-9 and gt-18, but by down-regulated DEGs in gt-17. Across all genotypes, analysis of DEG enzyme activities indicated an excess of down-regulated putative apoplastic peroxidases in the roots as water stress increased. This suggests that changes in root cell-wall architecture may be an important component of the response to water stress in Brachiaria