New chloroplast microsatellite markers suitable for assessing genetic diversity of Lolium perenne and other related grass species

Background and Aims

Lolium perenne (perennial ryegrass) is the most important forage grass species of temperate regions. We have previously released the chloroplast genome sequence of L. perenne ‘Cashel’. Here nine chloroplast microsatellite markers are published, which were designed based on knowledge about genetically variable regions within the L. perenne chloroplast genome. These markers were successfully used for characterizing the genetic diversity in Lolium and different grass species.

Methods

Chloroplast genomes of 14 Poaceae taxa were screened for mononucleotide microsatellite repeat regions and primers designed for their amplification from nine loci. The potential of these markers to assess genetic diversity was evaluated on a set of 16 Irish and 15 European L. perenne ecotypes, nine L. perenne cultivars, other Lolium taxa and other grass species.

Key Results

All analysed Poaceae chloroplast genomes contained more than 200 mononucleotide repeats (chloroplast simple sequence repeats, cpSSRs) of at least 7 bp in length, concentrated mainly in the large single copy region of the genome. Nucleotide composition varied considerably among subfamilies (with Pooideae biased towards poly A repeats). The nine new markers distinguish L. perenne from all non-Lolium taxa. TeaCpSSR28 was able to distinguish between all Lolium species and Lolium multiflorum due to an elongation of an A₈ mononucleotide repeat in L. multiflorum. TeaCpSSR31 detected a considerable degree of microsatellite length variation and single nucleotide polymorphism. TeaCpSSR27 revealed variation within some L. perenne accessions due to a 44-bp indel and was hence readily detected by simple agarose gel electrophoresis. Smaller insertion/deletion events or single nucleotide polymorphisms detected by these new markers could be visualized by polyacrylamide gel electrophoresis or DNA sequencing, respectively.

Conclusions

The new markers are a valuable tool for plant breeding companies, seed testing agencies and the wider scientific community due to their ability to monitor genetic diversity within breeding pools, to trace maternal inheritance and to distinguish closely related species.     

Effects of endophytic fungi on the phenotypic plasticity of Lolium perenne (Poaceae)

The effects of high vs. low levels of endophytic fungi on the phenotypic plasticity of cloned genotypes were examined in perennial ryegrass (Lolium perenne L.). The objectives were to determine whether endophytic fungi influence plastic responses of host genotypes to variable soil nutrients and whether or not endophyte infection and host genotype interact to determine the extent of this plasticity. Twelve infected genotypes were cloned into ramets: half the ramets were treated with the systemic fungicide Benomyl to reduce or eliminate the endophyte, while the other half were untreated. Ramets of each genotype were subjected to high, medium, or low levels of nutrients in the greenhouse for 11 wk. Tiller number, leaf area, and leaf mass were determined after 11 and 25 wk. The fungicide significantly reduced the level of endophyte infection. Responses to nutrient conditions in relation to fungicide treatment were genotype specific: for some genotypes, high levels of endophytic fungi appeared to reduce plasticity, while for other genotypes the endophyte had no effect. The potential for microscopic symbionts to affect phenotypic plasticity in genetically variable populations has not often been recognized. However, the clandestine effects of symbionts on the plasticity of host genotypes could impact microevolutionary processes occurring within plant populations that occupy heterogeneous environments.     

Development of a genomic microsatellite library in perennial ryegrass (Lolium perenne) and its use in trait mapping

Background and Aims: Perennial ryegrass (Lolium perenne) is one of the key forageand amenity grasses throughout the world. In the UK it accountsfor 70 % of all agricultural land use with an estimated farmgate value of £6 billion per annum. However, in termsof the genetic resources available, L. perenne has lagged behindother major crops in Poaceae. The aim of this project was thereforethe construction of a microsatellite-enriched genomic libraryfor L. perenne to increase the number of genetic markers availablefor both marker-assisted selection in breeding programmes andgene isolation. Methods: Primers for 229 non-redundant microsatellite markers were designedand used to screen two L. perenne genotypes, one amenity andone forage. Of the 229 microsatellites, 95 were found to showpolymorphism between amenity and forage genotypes. A selectionof microsatellite primers was selected from these 95 and usedto screen two mapping populations derived from intercrossingand backcrossing the two forage and amenity grass genotypes. Key Results and Conclusions: The utility of the resulting genetic maps for analysis of thegenetic control of target traits was demonstrated by the mappingof genes associated with heading date to linkage groups 4 and7.     

Universal primers for the amplification of chloroplast microsatellites in grasses (Poaceae)

Attempts to design truly universal primers to amplify chloroplast microsatellites have met with limited success due to nonconservation of repeat loci across widely divergent taxa. We have used the complete chloroplast genome sequences of rice, maize and wheat to design five pairs of primers that amplify homologous mononucleotide repeats across the Poaceae (grasses). Sequencing confirmed conservation of repeat motifs across subfamilies and a preliminary study in Anthoxanthum odoratum revealed polymorphism at two loci with a haplotype diversity value of 0.495. These primers provide a valuable tool to study cytoplasmic diversity in this extensively studied and economically important range of taxa.     

Development of novel microsatellite markers for the grassland species Lolium multiflorum,Lolium perenne and Festuca pratensis

Twelve microsatellite markers were isolated from Lolium multiflorum. Allelic variability and cross‐species amplification were assessed on 16 individuals of each of the three grassland species L. multiflorum, Lolium perenne and Festuca pratensis. Cross‐species amplification success was 100% for L. perenne and 83% for F. pratensis. The number of alleles detected ranged from one to 14 with an average of 3.4. While three microsatellite loci were polymorphic in all three species, one marker produced species‐specific alleles in all three species. These microsatellite markers provide a valuable tool for population genetic studies within and among species of the Festuca–Lolium complex.     

Cross-species amplification tests and diversity analysis using 56 PCR markers in Dactylis glomerata and Lolium perenne

We report results of cross-species amplification in Dactylis glomerata and Lolium perenne of 12 simple sequence repeats (SSRs) isolated from Lolium multiflorum×Festuca glaucescens, 42 SSRs from Festuca arundinacea and two sequence tagged sites from Oryza sativa. We compared the transferability and diversity between D. glomerata and L. perenne, which are important forage crops. While Nei's gene diversity values were equivalent in both species (from 0.14 to 0.92), the mean number of allele per locus was more important in D. glomerata than in L. perenne (5.45 vs. 4.50). These markers will be used for analysing population structure in grassland populations under agronomic practices.     

A high-density molecular map for ryegrass (Lolium perenne) using AFLP markers

AFLP markers have been successfully employed for the development of a high-density linkage map of ryegrass (Lolium perenne L.) using a progeny set of 95 plants from a testcross involving a doubled-haploid tester. This genetic map covered 930 cM in seven linkage groups and was based on 463 amplified fragment length polymorphism (AFLP) markers using 17 primer pairs, three isozymes and five EST markers. The average density of markers was approximately 1 per 2.0 cM. However, strong clustering of AFLP markers was observed at putative centromeric regions. Around these regions, 272 markers covered about 137 cM whereas the remaining 199 markers covered approximately 793 cM. Most genetic distances between consecutive pairs of markers were smaller than 20 cM except for five gaps on groups A, C, D, F and G. A skeletal map with a uniform distribution of markers can be extracted from this high-density map, and can be applied to detect and map QTLs. We report here the application of AFLP markers to genome mapping, in Lolium as a prelude to quantitative trait locus (QTL) identification for diverse agronomic traits in ryegrass and for marker-assisted plant breeding. Received: 4 November 1998 / Accepted:15 March 1999     

Development of novel chloroplast microsatellite markers for Miscanthus species (Poaceae)

? Premise of the study: A set of novel chloroplast microsatellite markers (cpSSRs) was developed for the bioenergy crop Miscanthus, and their utility in cross-species amplification was evaluated. ? Methods and Results: Twenty-eight novel primers flanking cpSSR loci were designed from a complete chloroplast genome sequence of Saccharum officinarum, a species closely related to Miscanthus. These primers were then tested on eight Miscanthus species, among which 16 cpSSR loci were found to be polymorphic. The number of alleles per polymorphic locus ranged from two to seven, with an average 3.94 alleles. ? Conclusions: These cpSSR markers can be applied to all Miscanthus species and will be useful for studying Miscanthus population structure, diversity, and phylogeography.     

Development and characterisation of simple sequence repeat (SSR) markers for perennial ryegrass (Lolium perenne L.)

Enrichment methods were optimised in order to isolate large numbers of simple sequence repeat (SSR) markers for perennial ryegrass (Lolium perenne L.), with the aim of developing a comprehensive set of loci for trait mapping and cultivar identification. Two libraries were constructed showing greater than 50% enrichment for a variety of SSR-motif types. Sequence characterisation of 1853 clones identified 859 SSR-containing clones, of which 718 were unique. Truncation of flanking sequences limited potential primer design to 366 clones. One-hundred selected SSR primer pairs were evaluated for amplification and genetic polymorphism across a panel of diverse genotypes. The efficiency of amplification was 81%. A relatively high level of SSR polymorphism was detected (67%), with a range of 2–7 alleles per locus. Mendelian segregation of alleles detected by selected SSR-locus primer pairs was demonstrated in the F₁ progeny of a pair cross. Cross-species amplification was detected in a number of related pasture and turfgrass species, with high levels of transfer to other Lolium species and members of the related genus Festuca. The identity of putative SSR ortholoci in these related species was confirmed by DNA sequence analysis. These loci constitute a valuable resource of ideal markers for the molecular breeding of ryegrasses and fescues. Received: 8 May 2000 / Accepted: 13 June 2000     

Isolation and characterization of novel microsatellite markers for Avena sativa (Poaceae) (oat)

? Premise of the study: A new set of microsatellite primers was developed for Avena sativa and characterized to assess the level of genetic diversity among cultivars and wild genotypes. ? Methods and Results: Using an enrichment genomic library, 14 simple sequence repeat markers were identified. The loci of these markers were characterized and found to be polymorphic in size among 48 genotypes of oat from diverse geographical locations. The number of alleles per locus ranged from two to eight, while the observed heterozygosity ranged from 0.031 to 0.75. ? Conclusions: These newly identified microsatellite markers will facilitate genetic diversity studies, fingerprinting, and genetic mapping of oat. Moreover, these new primers for A. sativa will aid future studies of polyploidy and hybridization in other species in this genus.     

Development and characterization of microsatellite markers for the tropical tree species Tabebuia aurea (Bignoniaceae)

In this study, we report the development and characterization of 21 polymorphic microsatellite loci for Tabebuia aurea, using genomic library enrichment. Number of alleles per locus and expected heterozygosity ranged from nine to 26 and from 0.808 to 0.955. The high combined probability of genetic identity (1.03 × 10^?37) and probability of paternity exclusion (0.9889) showed that multilocus genotypes are likely to be unique and will allow detailed parentage studies in natural populations of T. aurea. Additionally, a high percentage of transferability was achieved for the four species of the same genus studied.     

Repetitive flanking sequences (ReFS): novel molecular markers from microsatellite families

Although microsatellite markers have become exceedingly popular in molecular studies of wild organisms, their development in some taxonomic groups is challenging. This is partly because of repetitive flanking sequences, which lead to the simultaneous amplification of alleles from multiple loci. Until now, these microsatellite DNA families have been considered unsuitable for population genetics studies, but here we describe our development of these repetitive flanking sequences (ReFS) as novel molecular markers. We illustrate the utility of these markers by using them to address an outstanding taxonomic question in the moth genus Schrankia.     

Sixty simple sequence repeat markers for use in the Festuca–Lolium complex of grasses

So far only very few simple sequence repeat (SSR) markers developed from grass species have had their primer sequences published. To make more markers available to the scientific community, we isolated and sequenced 256 microsatellite‐containing clones from four genome libraries of a Lolium multiflorum×Festuca glaucescens F₁ hybrid following enrichment in (TC)_n, (TG)_n, or both repeats. In this work, we report the primer sequences of 60 SSRs including preliminary results of polymorphism for mapping.     

Osmoregulation and role of nitrate during regrowth after cutting of ryegrass (Lolium perenne)

The osmotic role of nitrate during aftermath growth of Lolium perenne L. cv. Réveille was investigated. Plants were grown from seed in a controlled environment using a liquid medium with 1.0 m M NH₄NO₃ as nitrogen source.
Eight-week-old plants were cut 4.0 cm above the root system and then harvested over a 14-day period of regrowth on the same initial nutrient solution, except that nitrate was ¹⁵N labelled. Throughout the experimental period, nitrate storage and reduction in roots were low. In stubble and especially in leaves, nitrate accumulated during the first 6 days of regrowth whereas nitrate reduction mainly occurred after this period. Analyses of carbohydrate, chloride and potassium contents in stubble and leaves showed that the accumulation of nitrate osmotically compensated for the decrease in soluble sugars during the first 6 days of regrowth.
The cumulative osmotic potential of sugars, chloride and nitrate in differently treated plants was studied in stubble and leaves. Compared with uncut plants, the lower carbohydrate concentrations found in cut plants regrowing on 1.0 m M NH₄NO₃ were compensated for by an accumulation of nitrate. During aftermath growth on low nitrogen nutrition (0.2 m M NH₄NO₃), chloride replaced nitrate, supporting the proposed osmotic function of nitrate.
It is concluded that nitrate is involved in the osmotic adjustment of ryegrass during regrowth after cutting.     

The effect of an experimental growth retardant (WL83801) on leaf growth and development in Lolium perenne L.

The experimental growth retardant WL83801, applied as a root drench, had a rapid and persistent effect in retarding the growth and development of leaves in L. perenne. Leaves of the main shoot were greatly reduced in length, were broader, and appeared faster than in control plants. The rate of extension of individual leaves was greatly reduced in retarded plants but still followed a diurnal pattern that closely corresponded with temperature. There was evidence that leaf extension was far less responsive to temperature in treated plants. At the cellular level WL83801 had no significant effect on leaf blade cell number, thus reductions in leaf length were associated with the retardation of cell elongation. Changes in leaf structure were also observed. These changes in the pattern of leaf growth and development are discussed in relation to the primary mode of action of the growth retardant in interfering with gibberellin biosynthesis.     

Ultrastructural features of a Stagonospora species (Ascomycotina) in senescent tissues of perennial ryegrass (Lolium perenne L.)

The pycnidium of a Stagonospora sp. observed in degenerating leaf sheath and blade tissues of Lolium perenne L. contained concentric bodies similar to those found in the mycobiont of several lichens and a few other free-living ascomycetes. These bodies occurred in vegetative hyphae, hyphae of the pycnidial wall, in conidiogenous cells and in conidia and comprised a central electron-translucent core surrounded by a granular zone and an outer radially orientated fibrillar or lamellar zone. In some cases the entire body was surrounded by a translucent halo. A second feature of the interwoven hyphae of the pycnidial wall was the occurrence of pores connecting adjacent hyphae. These lateral pores appeared identical to pores in septa between cells of individual hyphae, and like them, were associated with Woronin bodies.     

Genotyping by RAD sequencing enables mapping of fatty acid composition traits in perennial ryegrass (Lolium perenne (L.))

Perennial ryegrass (Lolium perenne L.) is the most important forage crop in temperate livestock agriculture. Its nutritional quality has significant impact on the quality of meat and milk for human consumption. Evidence suggests that higher energy content in forage can assist in reducing greenhouse gas emissions from ruminants. Increasing the fatty acid content (especially α‐linolenic acid, an omega‐3 fatty acid) may thus contribute to better forage, but little is known about the genetic basis of variation for this trait. To this end, quantitative trait loci (QTLs) were identified associated with major fatty acid content in perennial ryegrass using a population derived from a cross between the heterozygous and outbreeding high‐sugar grass variety AberMagic and an older variety, Aurora. A genetic map with 434 restriction‐associated DNA (RAD) and SSR markers was generated. Significant QTLs for the content of palmitic (C16:0) on linkage groups (LGs) 2 and 7; stearic (C18:0) on LGs 3, 4 and 7; linoleic (C18:2n‐6) on LGs 2 and 5; and α‐linolenic acids (C18:3n‐3) on LG 1 were identified. Two candidate genes (a lipase and a beta‐ketoacyl CoA synthase), both associated with C16:0, and separately with C18:2n‐6 and C18:0 contents, were identified. The physical positions of these genes in rice and their genetic positions in perennial ryegrass were consistent with established syntenic relationships between these two species. Validation of these associations is required, but the utility of RAD markers for rapid generation of genetic maps and QTL analysis has been demonstrated for fatty acid composition in a global forage crop.     

Plastome phylogenomics and phylogenetic diversity of endangered and threatened grassland species (Poaceae) in a North American tallgrass prairie

Native grasslands are one of the most endangered ecosystems in North America. In this study, we examined the ecological and evolutionary roles of endangered and threatened (e/t) grasses by establishing robust evolutionary relationships with other nonthreatened native and introduced grass species of the community. We hypothesized that the phylogenomic distribution of e/t species of grasses in Illinois would be phylogenetically clustered because closely related species would be vulnerable to the same threats and have similar requirements for survival. This study presents the first time a phylogeny based on complete plastome DNA of Poaceae was analyzed by phylogenetic diversity analysis. To avoid the disturbance of e/t populations, DNA was extracted from herbarium specimens. Next‐generation sequencing (NGS) techniques were used to sequence DNA of plastid genomes (plastomes). The resulting phylogenomic tree was analyzed by phylogenetic diversity metrics. The extracted DNA successfully produced complete plastomes demonstrating that herbarium material is a practical source of DNA for genomic studies. The phylogenomic tree was strongly supported and defined Dichanthelium as a separate clade from Panicum. The phylogenetic metrics revealed phylogenetic clustering of e/t species, confirming our hypothesis.