Assessing the validity and sensitivity of microbial processes within a hydrodynamic model.

Eutrophication due to excess anthropogenic nutrients in waterways is a significant issue worldwide. The pressure-stressor-response of a waterway to excessive nutrient loading is reliant on numerous physical and biological factors, including hydrodynamics and microbial processing. While substantial progress has been made towards simulating these mechanisms there are limited multi-disciplinary studies that relate the physical hydrodynamics of a site with the ecological response from linked laboratory and field studies. This paper presents the development of a coupled hydrodynamic and aquatic ecosystem response model, expanded to include an integrated microbial loop, that allows the explicit representation of heterotrophic bacteria growth and dissolved organic nutrient mineralisation. A unique long-term water quality dataset at an estuary in south-eastern Australia was used to validate and assess the model's sensitivity to complex biophysical processes driving the observed water quality variability. Results indicate that explicit time-varying bacterial mineralisation rates provide a substantially improved understanding of the broader aquatic ecosystem response than assigned fixed bulk rate parameter values, which are typically derived from non-local literature. Implementation of a microbial loop at the study site indicated that the model is sensitive to the boundary conditions, in particular catchment loads, with both net transport rates and the net growth rates of heterotrophic bacteria demonstrating different responses. Under average flow conditions, a smaller net transport and reduced nutrient availability has a pronounced effect of lowering net growth rates through the applied limitation factors. During high flow conditions, freshwater inflows increased net transport and nutrient loads, which resulted in higher net growth rates. Further, temporal variability in water temperature had a compounding effect on the model's response sensitivity. This approach has broader application in other riverine systems subject to eutrophication, and in interrogating linkages in hydrodynamic and microbial mediated processes (e.g., productivity). Future studies are recommended to better understand the sensitivity of aquatic ecosystem response models to microbial net growth rate kinetics at different temperatures and from top-down predation (e.g., zooplankton grazing).

Implications of bacterial mineralisation in aquatic ecosystem response models.

Widespread wastewater pollution is a major barrier to the sustainable management of freshwater and coastal marine ecosystems worldwide. Integrated multi-disciplinary studies are necessary to improve waterway management and protect ecosystem integrity. This study used the Generalised Likelihood Uncertainty Estimation (GLUE) methodology to link microbial community ecotoxicology laboratory data to a mechanistic aquatic ecosystem response model. The generic model provided good predictive skill for major water quality constituents, including heterotrophic bacteria dynamics (r2 = 0.91). The model was validated against observed data across a gradient of effluent concentrations from community whole effluent toxicity (WET) laboratory tests. GLUE analysis revealed that a combined likelihood measure increased confidence in the predictive capability of the model. This study highlights the importance of calibrating aquatic ecosystem response models with net growth rates (i.e., sum of the growth minus loss rate parameter terms) of biological functional groups. The final calibrated net growth rate value of heterotrophic bacteria determined using the GLUE analysis was selected to be 0.58, which was significantly greater than the average literature value of -0.15. This finding demonstrated that use of literature parameter values without a good understanding of the represented processes could create misleading outputs and result in unsatisfactory conclusions. Further, fixed bulk mineralisation rate literature values are typically higher than realistically required in aquatic ecosystem response models. This indicates that explicitly including bacterial mineralisation is crucial to represent microbial ecosystem functioning more accurately. Our study suggests that improved data collection and modelling efforts in real-world management applications are needed to better address nutrients released into the natural environment. Future studies should aim to better understand the sensitivity of aquatic ecosystem response models to bacterial mineralisation rates.

Identifying genetic components controlling fertility in the outcrossing grass species perennial ryegrass (Lolium perenne) by quantitative trait loci analysis and comparative genetics.

Mutational load and resource allocation factors and their effects on limiting seed set were investigated in ryegrass by comparative mapping genomics and quantitative trait loci (QTL) analysis in two perennial ryegrass (Lolium perenne) mapping families sharing common genetic markers. Quantitative trait loci for seed-set were identified on chromosome (LG) 7 in both families and on LG4 of the F2/WSC family. On LG7, seed-set and heading date QTLs colocalized in both families and cannot be unequivocally resolved. Comparative genomics suggests that the LG7 region is syntenous to a region of rice LG6 which contains both fertility (S5(n)) and heading date (Hd1, Hd3a) candidate genes. The LG4 region is syntenous to a region of rice LG3 which contains a fertility (S33) candidate gene. QTL maxima for seed-set and heading date on LG4 in the F2/WSC family are separated by c. 8 cm, indicating distinct genetic control. Low seed set is under the control of recessive genes at both LG4 and LG7 locations. The identification of QTLs associated with seed set, a major component of seed yield in perennial ryegrass, indicates that mutational load associated with these genomic regions can be mitigated through marker-assisted selection.

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 forage and amenity grasses throughout the world. In the UK it accounts for 70 % of all agricultural land use with an estimated farm gate value of 6 billion pounds per annum. However, in terms of the genetic resources available, L. perenne has lagged behind other major crops in Poaceae. The aim of this project was therefore the construction of a microsatellite-enriched genomic library for L. perenne to increase the number of genetic markers available for both marker-assisted selection in breeding programmes and gene isolation. METHODS: Primers for 229 non-redundant microsatellite markers were designed and used to screen two L. perenne genotypes, one amenity and one forage. Of the 229 microsatellites, 95 were found to show polymorphism between amenity and forage genotypes. A selection of microsatellite primers was selected from these 95 and used to screen two mapping populations derived from intercrossing and backcrossing the two forage and amenity grass genotypes. KEY RESULTS AND CONCLUSIONS: The utility of the resulting genetic maps for analysis of the genetic control of target traits was demonstrated by the mapping of genes associated with heading date to linkage groups 4 and 7.

Identification of perennial ryegrass (Lolium perenne (L.)) and meadow fescue (Festuca pratensis (Huds.)) candidate orthologous sequences to the rice Hd1(Se1) and barley HvCO1 CONSTANS-like genes through comparative mapping and microsynteny.

Microsynteny with rice and comparative genetic mapping were used to identify candidate orthologous sequences to the rice Hd1(Se1) gene in Lolium perenne and Festuca pratensis. A F. pratensis bacterial artificial chromosome (BAC) library was screened with a marker (S2539) physically close to Hd1 in rice to identify the equivalent genomic region in F. pratensis. The BAC sequence was used to identify and map the same region in L. perenne. Predicted protein sequences for L. perenne and F. pratensis Hd1 candidates (LpHd1 and FpHd1) indicated they were CONSTANS-like zinc finger proteins with 61-62% sequence identity with rice Hd1 and 72% identity with barley HvCO1. LpHd1 and FpHd1 were physically linked in their respective genomes (< 4 kb) to marker S2539, which was mapped to L. perenne chromosome 7. The identified candidate orthologues of rice Hd1 and barley HvCO1 in L. perenne and F. pratensis map to chromosome 7, a region of the L. perenne genome which has a degree of conserved genetic synteny both with rice chromosome 6, which contains Hd1, and barley chromosome 7H, which contains HvCO1.

Molecular tagging of a senescence gene by introgression mapping of a stay-green mutation from Festuca pratensis.

* Intergeneric hybrids between Lolium multiflorum and Festuca pratensis (Lm/Fp) and their derivatives exhibit a unique combination of genetic and cytogenetic characteristics: chromosomes undergo a high frequency of homoeologous recombination at meiosis; the chromosomes of the two species can easily be discriminated by genomic in situ hybridization (GISH); recombination occurs along the entire length of homoeologous bivalents; a high frequency of marker polymorphism is observed between the two species. * This combination of characters has been used to transfer and isolate a F. pratensis chromosome segment carrying a mutant 'stay-green' gene conferring a disrupted leaf senescence phenotype into L. multiflorum. * The genetic location within the introgressed F. pratensis segment of the senescence gene has been mapped using amplified fragment length polymorphisms (AFLPs), and F. pratensis-specific AFLP markers closely flanking the green gene have been cloned. * The use of these cloned sequences as markers for the stay-green locus in marker-assisted selection programmes has been tested. The potential application of Lm/Fp introgressions as a tool for the map-based cloning of introgressed Fp genes is discussed.

Synteny between a major heading-date QTL in perennial ryegrass (Lolium perenne L.) and the Hd3 heading-date locus in rice.

The genetic control of induction to flowering has been studied extensively in both model and crop species because of its fundamental biological and economic significance. An ultimate aim of many of these studies has been the application of the understanding of control of flowering that can be gained from the study of model species, to the improvement of crop species. The present study identifies a region of genetic synteny between rice and Lolium perenne, which contains the Hd3 heading-date QTL in rice and a major QTL, accounting for up to 70% of the variance associated with heading date in L. perenne. The identification of synteny between rice and L. perenne in this region demonstrates the direct applicability of the rice genome to the understanding of biological processes in other species. Specifically, this syntenic relationship will greatly facilitate the genetic dissection of aspects of heading-date induction by enabling the magnitude of the genetic component of the heading-date QTL in L. perenne to be combined with the sequencing and annotation information from the rice genome.

A demonstration of a 1:1 correspondence between chiasma frequency and recombination using a Lolium perenne/Festuca pratensis substitution.

A single chromosome of the grass species Festuca pratensis has been introgressed into Lolium perenne to produce a diploid monosomic substitution line 2n = 2x = 14. The chromatin of F. pratensis and L. perenne can be distinguished by genomic in situ hybridization (GISH), and it is therefore possible to visualize the substituted F. pratensis chromosome in the L. perenne background and to study chiasma formation in a single marked bivalent. Recombination occurs freely in the F. pratensis/L. perenne bivalent, and chiasma frequency counts give a predicted map length for this bivalent of 76 cM. The substituted F. pratensis chromosome was also mapped with 104 EcoRI/Tru91 and HindIII/Tru91 amplified fragment length polymorphisms (AFLPs), generating a marker map of 81 cM. This map length is almost identical to the map length of 76 cM predicted from the chiasma frequency data. The work demonstrates a 1:1 correspondence between chiasma frequency and recombination and, in addition, the absence of chromatid interference across the Festuca and Lolium centromeres.

Physical and genetic mapping in the grasses Lolium perenne and Festuca pratensis.

A single chromosome of the grass species Festuca pratensis has been introgressed into Lolium perenne to produce a diploid monosomic substitution line 2n = 2x = 14. In this line recombination occurs throughout the length of the F. pratensis/L. perenne bivalent. The F. pratensis chromosome and recombinants between it and its L. perenne homeologue can be visualized using genomic in situ hybridization (GISH). GISH junctions represent the physical locations of sites of recombination, enabling a range of recombinant chromosomes to be used for physical mapping of the introgressed F. pratensis chromosome. The physical map, in conjunction with a genetic map composed of 104 F. pratensis-specific amplified fragment length polymorphisms (AFLPs), demonstrated: (1) the first large-scale analysis of the physical distribution of AFLPs; (2) variation in the relationship between genetic and physical distance from one part of the F. pratensis chromosome to another (e.g., variation was observed between and within chromosome arms); (3) that nucleolar organizer regions (NORs) and centromeres greatly reduce recombination; (4) that coding sequences are present close to the centromere and NORs in areas of low recombination in plant species with large genomes; and (5) apparent complete synteny between the F. pratensis chromosome and rice chromosome 1.

5-azacytidine induces chromosomal breakage in the root tips of wheat carrying the cuckoo chromosome 4S(L) from Aegilops sharonensis.

The cuckoo chromosome 4S(L) from Aegilops sharonensis is preferentially transmitted when introduced by hybridization into common wheat, Triticum aestivum. Gametocidal (Gc) factors carried in 4S(L) induce chromosome breakage in meiospores not containing them, ensuring their transmission to the progeny. Chromosome breakage and break-fusion-bridge (BFB) cycles can also be observed during early embryo sac development of chromosome 4S(L) addition lines to wheat, often leading to the presence of dicentric chromosomes in the subsequent progeny. However, the process responsible for inducing the primary chromosomal breaks only appears to occur during the initial divisions of the embryo and endosperm. In the presence of chromosome 4S(L), treatment with the hypomethylating agent 5-azacytidine induces chromosome breakage in root tips. This suggests that the process of chromosome fragmentation, induced by the Gc factors during early seed development, is repressed at later stages by DNA methylation.

Genetic and physical analysis of a single Festuca pratensis chromosome segment substitution in Lolium perenne.

Molecular marker analysis and genomic in situ hybridisation (GISH) were used to examine the process of chromosome segment introgression in BC2 diploid hybrids (2n=2x=14) between Lolium perenne and Festuca pratensis. Two genotypes having what appeared to be the same, single, introgressed chromosome segment of F. pratensis in the L. perenne background were crossed with diploid L. perenne to produce a recombinant series for the introgressed region. Physical and genetic analysis of this series showed that, while recombination seemed to be possible at all points along the chromosome arm, the rate of recombination varied depending on relative position: more recombination was detected in the interstitial region as compared with the centromeric or telomeric regions. The implications of these results for the use of GISH and molecular marker analysis in the measurement of linkage drag in backcross breeding programmes is discussed.

Chromosome pairing in Lolium perenne x L. temulentum diploid hybrids: genetic and cytogenetic evaluation.

A Lolium perenne genotype (E5/2/5/10), which had been selected for low chiasma frequency over a number of generations and which was suspected of containing one or two heterozygous dominant genes with a significant effect on chiasma frequency, was crossed with L. temulentum (Ba3081) to create a hybrid population of 47 diploid plants. The mean chiasma or paired arm (PA) frequency of homoeologous chromosomes at meiosis in the population was 9.1/cell (1.3 PA/chromosome pair) with a distribution skewed towards high PA frequency. More than 90% of the hybrid chromosomes paired at meiosis in spite of the disparity in chromosome length and DNA quantity between the two species. Overall, the distribution of PAs between chromosomes for a given number of PAs/cell favoured the production of rod bivalents over ring bivalents and univalents, indicating that there is a mechanism present that maximizes the total number of bivalent associations formed. Molecular marker analysis using AFLPs and isoenzymes did not identify any clear major gene effect on PA frequency in the hybrid population. It was concluded that the control of PA frequency in E5/2/5/10 was not a simple genetic mechanism.

Physical mapping of ribosomal DNA sites in Festuca arundinacea and related species by in situ hybridization.

UNLABELLED: The positions of the 18S-5.8S-26S and 5S rRNA genes have been physically mapped on the chromosomes of diploid, tetraploid, and hexaploid Festuca species by in situ hybridization. The number and position of the rDNA sites in the species were compared. The results confirm some of the earlier phylogenetic studies of these species but suggest that some structural rearrangements have occurred and that sites have been lost during polyploidization. KEYWORDS: Festuca, in situ hybridization, phylogeny, physical mapping, rDNA.

Comparison of ribosomal DNA sites in Lolium species by fluorescence in situ hybridization.

The position of the 18S-5.8S-26S and 5S rRNA genes have been physically mapped on the chromosomes of seven Lolium taxa. 18S-5.8S-26S sites were seen on two pairs of chromosomes in the inbreeding taxa. In the outbreeding taxa six sites were found in the L. multiflorum, seven in L. perenne and nine in L. rigidum var. rigidum. Two 5S sites were found in each of the taxa. In the inbreeders, the 5S sites were found adjacent to the 18S-5.8S-26S sites on chromosome 2. In L. multifiorum and L.perenne the 5S sites were on the short arm of chromosome 3. However, in L. rigidum var. rigidum the 5S rDNA site was found in either of the two positions.

Detection of interchromosomal translocations within the Triticeae by RFLP analysis.

Twenty-three wheat/alien addition or substitution lines were screened using restriction fragment length polymorphisms for the presence or absence of 4/5 and 4/7 reciprocal translocations in the alien chromosomes. Such translocations have previously been identified in wheat and rye. Group 4 and group 5 Aegilops umbellulata, Triticum urartu, and Thinopyrum bessarabicum chromosomes were found to carry 4/5 translocations. Evidence for a 4/7 translocation was also found in Secale montanum. The presence of the 4/5 translocations in T. urartu indicates that the translocation predates the polyploidization of wheat. The implications of these results are discussed.

Detection of ribosomal DNA sites in lentil and chickpea by fluorescent in situ hybridization.

Hybridization sites of an rDNA probe coding for the 18S, 5.8S, and 26S genes were detected on lentil and chickpea somatic chromosomes using fluorescent in situ hybridization. One pair of hybridization sites was detected in cultivated lentil Lens culinaris L. and wild lentil L. orientalis (Boiss.) Hand.-Mazz., and in both the hybridization sites of the ribosomal probe correspond to the secondary constriction. In cultivated chickpea Cicer arietinum three pairs of rDNA sites were detected and in the wild C. reticulatum two pairs were detected. The karyotypic relationship between the cultivated C. arietinum and its wild progenitor C. reticulatum is discussed.

Intergenomic translocations and the genomic composition of Avena maroccana Gdgr. revealed by FISH.

Fluorescence in situ hybridization (FISH) using total genomic DNA from putative diploid progenitors was used to confirm the presence of the A and C genomes in Avena maroccana. These results confirm cytological data that intergenomic translocations are present in A. maroccana.

Determination of the frequency of wheat-rye chromosome pairing in wheat x rye hybrids with and without chromosome 5B.

Genomic in-situ hybridization (GISH) was used to determine the amount of wheat-rye chromosome pairing in wheat (Triticum aestivum) x rye (Secale cereale) hybrids having chromosome 5B present, absent, or replaced by an extra dose of chromosome 5D. The levels of overall chromosome pairing were similar to those reported earlier but the levels of wheat-rye pairing were higher than earlier determinations using C-banding. Significant differences in chromosome pairing were found between the three genotypes studied. Both of the chromosome-5B-deficient hybrid genotypes showed much higher pairing than the euploid wheat hybrid. However, the 5B-deficient hybrid carrying an extra chromosome 5D had significantly less wheat-rye pairing than the simple 5B-deficient genotype, indicating the presence of a suppressing factor on chromosome 5D. Non-homologous/non-homoeologous chromosome pairing was observed in all three hybrid genotypes. The value of GISH for assessing the level of wheat-alien chromosome pairing in wheat/alien hybrids and the effectiveness of wheat genotypes that affect homoeologous chromosome pairing is demonstrated.

Primer-mediated in situ detection of the B-hordein gene cluster on barley chromosome 1H.

In situ hybridization methods allow the detection of specific DNA sequences on whole chromosomes. The technique has been widely used as a diagnostic and research tool by animal cytogeneticists, for whom detection of unique sequences on mammalian chromosomes is routinely achieved. However, detection of unique sequences on plant chromosomes is less reliable. The recently developed primer-induced in situ hybridization (PRINS) technique allows rapid and reliable in situ detection by the hybridization of primers to denatured target DNA, followed by extension with DNA polymerase in the presence of a labeled nucleotide. The use of short oligonucleotide primers could allow improved penetration of debris and highly condensed chromatin common in preparations of plant chromosomes, thus increasing the sensitivity of in situ detection. The feasibility of this approach is demonstrated by the oligonucleotide primer-mediated detection of the B-hordein gene cluster on a barley chromosome. Applications of the PRINS technique for plant cytogeneticists are discussed.