Susanne Barth

Heterosis for biomass yield and related traits in five hybrids of Arabidopsis thaliana L. Heynh

Heterosis is of utmost economic importance in plant breeding. However, its underlying molecular causes are still unknown. Given the numerous advantages of Arabidopsis thaliana as a model species in plant genetics and genomics, we assessed the extent of heterosis in this species using five hybrids derived from five ecotypes. Parents, F1 and F2, generations in both reciprocal forms were grown in a greenhouse experiment with four replications. Mid-parent heterosis (MPH) and best-parent heterosis (BPH) averaged across hybrids were surprisingly high for biomass yield (MPH: 60.3%; BPH: 32.9%) and rosette diameter (MPH: 49.4%; BPH: 34.8%), but smaller for flowering date (MPH: 27.5%; BPH: 18.5%), seed yield (MPH: 18.9%; BPH: 1.7%), and yield components. Individual hybrids varied considerably in their MPH and BPH values for all traits, one cross displaying 140.1% MPH for biomass yield. MPH was not associated with parental genetic distance determined from molecular markers. Reciprocal effects were significant only in a few cases. With a proper choice of hybrids, our results encourage the use of Arabidopsis as a model species for investigating the molecular causes of heterosis.

Transcriptional and metabolic profiles of Lolium perenne L. genotypes in response to a PEG‐induced water stress

Metabolic profiling was carried out in the forage grass Lolium perenne L. (perennial ryegrass) to uncover mechanisms involved in the plants response to water stress. When leaf and root materials from two genotypes, with a contrasting water stress response, were analysed by GC-MS, a clear difference in the metabolic profiles of the leaf tissue under water stress was observed. Differences were principally due to a reduction in fatty acid levels in the more susceptible Cashel genotype and an increase in sugars and compatible solutes in the more tolerant PI 462336 genotype. Sugars with a significant increase included: raffinose, trehalose, glucose, fructose and maltose. Increasing the ability of perennial ryegrass to accumulate these sugars in response to a water deficit may lead to more tolerant varieties. The metabolomics approach was combined with a transcriptomics approach in the water stress tolerant genotype PI 462336, which has identified perennial ryegrass genes regulated under water stress.

Analysis of a triple testcross design with recombinant inbred lines reveals a significant role of epistasis in heterosis for biomass-related traits in Arabidopsis

Primary causes of heterosis are still unknown. Our goal was to investigate the extent and underlying genetic causes of heterosis for five biomass-related traits in Arabidopsis thaliana. We (i) investigated the relative contribution of dominance and epistatic effects to heterosis in the hybrid C24 × Col-0 by generation means analysis and estimates of variance components based on a triple testcross (TTC) design with recombinant inbred lines (RILs), (ii) estimated the average degree of dominance, and (iii) examined the importance of reciprocal and maternal effects in this cross. In total, 234 RILs were crossed to parental lines and their F1s. Midparent heterosis (MPH) was high for rosette diameter at 22 days after sowing (DAS) and 29 DAS, growth rate (GR), and biomass yield (BY). Using the F2-metric, directional dominance prevailed for the majority of traits studied but reciprocal and maternal effects were not significant. Additive and dominance variances were significant for all traits. Additive × additive and dominance × dominance variances were significant for all traits but GR. We conclude that dominance as well as digenic and possibly higher-order epistatic effects play an important role in heterosis for biomass-related traits. Our results encourage the use of Arabidopsis hybrid C24 × Col-0 for identification and description of quantitative trait loci (QTL) for heterosis for biomass-related traits and further genomic studies.

Complete chloroplast genome sequence of a major allogamous forage species, perennial ryegrass (Lolium perenne L.).

Lolium perenne L. (perennial ryegrass) is globally one of the most important forage and grassland crops. We sequenced the chloroplast (cp) genome of Lolium perenne cultivar Cashel. The L. perenne cp genome is 135 282 bp with a typical quadripartite structure. It contains genes for 76 unique proteins, 30 tRNAs and four rRNAs. As in other grasses, the genes accD, ycf1 and ycf2 are absent. The genome is of average size within its subfamily Pooideae and of medium size within the Poaceae. Genome size differences are mainly due to length variations in non-coding regions. However, considerable length differences of 1–27 codons in comparison of L. perenne to other Poaceae and 1–68 codons among all Poaceae were also detected. Within the cp genome of this outcrossing cultivar, 10 insertion/deletion polymorphisms and 40 single nucleotide polymorphisms were detected. Two of the polymorphisms involve tiny inversions within hairpin structures. By comparing the genome sequence with RT–PCR products of transcripts for 33 genes, 31 mRNA editing sites were identified, five of them unique to Lolium. The cp genome sequence of L. perenne is available under Accession number AM777385 at the European Molecular Biology Laboratory, National Center for Biotechnology Information and DNA DataBank of Japan.

EST-derived SSR markers used as anchor loci for the construction of a consensus linkage map in ryegrass ( Lolium spp.)

BackgroundGenetic markers and linkage mapping are basic prerequisites for marker-assisted selection and map-based cloning. In the case of the key grassland species Lolium spp., numerous mapping populations have been developed and characterised for various traits. Although some genetic linkage maps of these populations have been aligned with each other using publicly available DNA markers, the number of common markers among genetic maps is still low, limiting the ability to compare candidate gene and QTL locations across germplasm.ResultsA set of 204 expressed sequence tag (EST)-derived simple sequence repeat (SSR) markers has been assigned to map positions using eight different ryegrass mapping populations. Marker properties of a subset of 64 EST-SSRs were assessed in six to eight individuals of each mapping population and revealed 83% of the markers to be polymorphic in at least one population and an average number of alleles of 4.88. EST-SSR markers polymorphic in multiple populations served as anchor markers and allowed the construction of the first comprehensive consensus map for ryegrass. The integrated map was complemented with 97 SSRs from previously published linkage maps and finally contained 284 EST-derived and genomic SSR markers. The total map length was 742 centiMorgan (cM), ranging for individual chromosomes from 70 cM of linkage group (LG) 6 to 171 cM of LG 2.ConclusionsThe consensus linkage map for ryegrass based on eight mapping populations and constructed using a large set of publicly available Lolium EST-SSRs mapped for the first time together with previously mapped SSR markers will allow for consolidating existing mapping and QTL information in ryegrass. Map and markers presented here will prove to be an asset in the development for both molecular breeding of ryegrass as well as comparative genetics and genomics within grass species.

How far are we from unravelling self‐incompatibility in grasses?

The genetic and physiological mechanisms involved in limiting self-fertilization in angiosperms, referred to as self-incompatibility (SI), have significant effects on population structure and have potential diversification and evolutionary consequences. Up to now, details of the underlying genetic control and physiological basis of SI have been elucidated in two different gametophytic SI (GSI) systems, the S-RNase SI and the Papaver SI systems, and the sporophytic SI (SSI) system (Brassica). In the grass family (Poaceae), which contains all the cereal and major forage crops, SI has been known for half a century to be controlled gametophytically by two multiallelic and independent loci, S and Z. But still none of the gene products for S and Z is known and only limited information on related biochemical responses is available. Here we compare current knowledge of grass SI with that of other well-characterized SI systems and speculate about the relationship between SSI and grass SI. Additionally, we discuss comparative mapping as a tool for the further investigation of grass SI.

An Optimized Chloroplast DNA Extraction Protocol for Grasses (Poaceae) Proves Suitable for Whole Plastid Genome Sequencing and SNP Detection

Background Obtaining chloroplast genome sequences is important to increase the knowledge about the fundamental biology of plastids, to understand evolutionary and ecological processes in the evolution of plants, to develop biotechnological applications (e.g. plastid engineering) and to improve the efficiency of breeding schemes. Extraction of pure chloroplast DNA is required for efficient sequencing of chloroplast genomes. Unfortunately, most protocols for extracting chloroplast DNA were developed for eudicots and do not produce sufficiently pure yields for a shotgun sequencing approach of whole plastid genomes from the monocot grasses. Methodology/Principal Findings We have developed a simple and inexpensive method to obtain chloroplast DNA from grass species by modifying and extending protocols optimized for the use in eudicots. Many protocols for extracting chloroplast DNA require an ultracentrifugation step to efficiently separate chloroplast DNA from nuclear DNA. The developed method uses two more centrifugation steps than previously reported protocols and does not require an ultracentrifuge. Conclusions/Significance The described method delivered chloroplast DNA of very high quality from two grass species belonging to highly different taxonomic subfamilies within the grass family (Lolium perenne, Pooideae; Miscanthus×giganteus, Panicoideae). The DNA from Lolium perenne was used for whole chloroplast genome sequencing and detection of SNPs. The sequence is publicly available on EMBL/GenBank.

Progress towards elucidating the mechanisms of self-incompatibility in the grasses: further insights from studies in Lolium

BACKGROUND AND SCOPE Self-incompatibility (SI) in flowering plants ensures the maintenance of genetic diversity by ensuring outbreeding. Different genetic and mechanistic systems of SI among flowering plants suggest either multiple origins of SI or considerable evolutionary diversification. In the grasses, SI is based on two loci, S and Z, which are both polyallelic: an incompatible reaction occurs only if both S and Z alleles are matched in individual pollen with alleles of the pistil on which they alight. Such incompatibility is referred to as gametophytic SI (GSI). The mechanics of grass GSI is poorly understood relative to the well-characterized S-RNase-based single-locus GSI systems (Solanaceae, Rosaceae, Plantaginaceae), or the Papaver recognition system that triggers a calcium-dependent signalling network culminating in programmed cell death. There is every reason to suggest that the grass SI system represents yet another mechanism of SI. S and Z loci have been mapped using isozymes to linkage groups C1 and C2 of the Triticeae consensus maps in Secale, Phalaris and Lolium. Recently, in Lolium perenne, in order to finely map and identify S and Z, more closely spaced markers have been developed based on cDNA and repeat DNA sequences, in part from genomic regions syntenic between the grasses. Several genes tightly linked to the S and Z loci were identified, but so far no convincing candidate has emerged. RESEARCH AND PROGRESS From subtracted Lolium immature stigma cDNA libraries derived from S and Z genotyped individuals enriched for SI potential component genes, kinase enzyme domains, a calmodulin-dependent kinase and a peptide with several calcium (Ca(2+)) binding domains were identified. Preliminary findings suggest that Ca(2+) signalling and phosphorylation may be involved in Lolium GSI. This is supported by the inhibition of Lolium SI by Ca(2+) channel blockers lanthanum (La(3+)) and verapamil, and by findings of increased phosphorylation activity during an SI response.

Expressed sequence tag-derived microsatellite markers of perennial ryegrass (Lolium perenne L.)

An expressed sequence tag (EST) library of the key grassland species perennial ryegrass (Lolium perenne L.) has been exploited as a resource for microsatellite marker development. Out of 955 simple sequence repeat (SSR) containing ESTs, 744 were used for primer design. Primer amplification was tested in eight genotypes of L. perenne and L. multiflorum representing (grand-) parents of four mapping populations and resulted in 464 successfully amplified EST-SSRs. Three hundred and six primer pairs successfully amplified products in the mapping population VrnA derived from two of the eight genotypes included in the original screening and revealed SSR polymorphisms for 143 ESTs. Here, we report on 464 EST-derived SSR primer sequences of perennial ryegrass established in laboratory assays, providing a dedicated tool for marker assisted breeding and comparative mapping within and among forage and turf grasses.

Chloroplast DNA markers (cpSSRs, SNPs) for Miscanthus, Saccharum and related grasses (Panicoideae, Poaceae)

Miscanthus and Saccharum are closely related perennial C4 grasses. Miscanthus has recently attracted interest as a non-food crop for energy and fibre production. However, molecular genetic tools for the selection of new Miscanthus genotypes and study of its genetic resources are limited. We have identified six chloroplast (plastid) marker loci,containing both microsatellites (cpSSRs) and single nucleotide polymorphisms (SNPs) and developed primers to amplify and sequence these regions. The primers were designed using the complete chloroplast genome sequence of sugarcane and were tested on a collection of 164 Miscanthus genotypes and 14 related species of the subfamily Panicoideae. The cpSSR markers were highly polymorphic, with the number of alleles ranging from 10 to 16 per locus. Within the six cpSSR marker loci, the hybrid M. ×giganteus exhibits virtually no cpDNA variation compared with its putative parents M. sinensis and M. sacchariflorus. These SNP markers enable the differentiation of most Miscanthus species and detect infraspecific variation suitable for defining cytoplasmic genepools of Miscanthus for breeding purposes.