The papers in this volume form the core of a meeting called Putting Plant Physiology on the Map, which took place at the University of Wales Bangor, in April 1997. The idea for the meeting came from Professor Sid Thomas, and the scientific organization was done by him and his colleagues at the Institute for Grassland and Environmental Research, Aberystwyth. The aim was to convene a small group of physiologists and geneticists who are concerned with the genetic mapping of plant physiological traits, to produce a meeting and published volume which would summarize some of the methods and results of existing mapping programmes, and more importantly to proselytize by laying out for unconverted plant physiologists the merits of taking a genetical approach. Writing as one of the unconverted, the meeting succeeded admirably: the power and utility of a genetical approach became very clear. Not only does mapping provide unequivocal answers to a range of physiological questions, it also enables one to ask questions that simply cannot be addressed by conventional physiological approaches. The progress which has been made in understanding salt tolerance, for example, adds a completely new dimension to the experiments in the classical literature.

Those with experience of putting plant physiological characteristics onto genetical maps inevitably have a language of their own, and for the newcomer the acquisition of this language is a daunting task. Since conversion was one of our aims, we have provided a chapter (Jones, Ougham & Thomas) which explains to the beginner just how maps are made, and in so doing hopefully eases the problem of acquiring the language. Virgin mappers should read this chapter first; the experienced can move straight to the rest and sample the variety of approaches which have been used.

The use of genetics in plant biology aims at the physiological and molecular genetical characterization of the phenotypic variation for the trait under study. Efficient mutant and gene isolation procedures have been developed for a number of plant models such as Arabidopsis thaliana. For this, the map position of the genes and insertion mutagenesis are used. The latter also allows the characterization of genes that are not easily recognized in mutant approaches, by using enhancer or gene-trapping procedures and reverse genetics. In addition to mutants, natural variation present among wild and cultivated varieties of a species provides an important source of genetic variation. The use of molecular markers, advanced mapping populations and specific cytogenetic stocks in case of polyploids, enables a detailed characterization of such natural variation even when it is of a quantitative and polygenic nature. Examples of the various genetic approaches are given.

Microseris douglasii (DC.) Sch.-Bip. and M. bigelovii (Gray) Sch.-Bip. are two small annual autogamous species of Compositae with nearly non-overlapping distribution ranges in Western North America. Specifically, M. bigelovii occurs directly along the Pacific coast, whilst M. douglasii has an inland distribution including patches of serpentine soil. Both species are variable, and artificial hybrids between them vary widely in fertility depending on the individual parents. Segregating offspring of one hybrid (strain H27) is being used to analyse the genetic basis of characters differentiating the two species by QTL mapping with RAPDS and ALFPs as molecular markers. Technical problems with mapping dominant markers in a wide cross will be briefly listed and QTL analysis will be discussed. For the genetic analysis of physiological characters, the precise definition of the characters is crucial and the methods of scoring or measuring phenotypes in different environments eventually require more time and effort than the molecular characterization. We are establishing recombinant inbred lines to provide material for more complex physiological analyses requiring several plants per genotype. An increasing number of characters is being studied in this cross, and the possibility of shared pleiotropic QTLs is high. The potential number of QTL genotypes by far exceeds the number of actual genotypes in these lines. We are characterizing the gene interactions as closely as possible and making quantitative genetic models to predict the genotypes corresponding to all possible genotypes. These predictions are converted via computer modelling into an increasingly realistic three-dimensional representation of the growing plant useful for a simulation of plant evolution.

The present understanding of the genetic control of flowering in wheat is reviewed. Although this information will continue to be of value in breeding and in recognizing the role such genes have had in adapting the wheat crop to global agriculture, its use in defining the processes responsible for flowering will be limited. Progress here will depend on developments in model plant systems such as Arabidopsis, where methods for isolating genes are much more advanced than in wheat. These developments, which have already started, will probably be used to establish homologies between the genes for flowering within the cereals, as well as more widely. An understanding of the flowering process in wheat is likely to emerge from this approach. In the meantime, there are some unknowns in the genetics of flowering in wheat which need to be resolved. These include the identification of a gene(s) on the group 4 chromosomes of wheat which is homoeoallelic with the gene Sh in barley. Also, the proposed gene(s) delaying flowering and located on the group 6 chromosomes needs to be recognized and mapped. Similar needs occur for the group 1 chromosomes as well as the resolution of whether or not Vrn5 is correctly positioned on chromosome 7BS.

A mutation of a gene (discovered in Festuca pratensis Huds. and designated sid ) confers indefinite greenness on senescing leaves. Via intergeneric hybrids with Lolium multiflorum L. and Lolium perenne L., the mutant gene (sidy) has been introgressed into a range of Lolium backgrounds. Using genomic in situ hybridization we have identified segments carrying sidy in recombinant chromosomes of Lolium–Festuca introgression lines. We also used L. perenne lines segregating 1[ratio ]1 for the staygreen character to tag the gene with molecular markers. In two mapping populations a total of 84 genotypes were screened with isoenzymes, RAPD primers, RFLP probes and AFLP primer pairs. Over 180 polymorphic loci were identified, representing 10 linkage groups spanning 600 cM. Two AFLP markers are linked to sid at 4·6 and 14·9 cM, close enough to be usable for marker-assisted selection. Introgression of sidy into Lolium temulentum L. resulted in the production of near-isogenic inbreeding lines suitable for comparative studies of gene expression. Using a variation of the method of representational difference analysis a very small number of cDNAs have been identified as promising candidates for sid, or genes directly regulated by sid.

Multiple-marker techniques based on PCR are highly informative in genetic analysis. The bulk of many genomes is comprised of repeated sequences, and thus anonymous markers derived from genomic DNA are likely to correspond to repeated DNA sequences. Here we show that repeated-sequence-derived markers can provide information similar in quality to that of other PCR-based markers. Presumably this reflects the abundance of this repeat type in anonymous PCR markers such as RAPDs or AFLPs.

The concept of the ‘nucleotype’ embraces aspects of the nuclear DNA that affect the phenotype other than through the coding sequences. In this context, we consider one particular situation, namely DNA variation due to the presence of supernumerary B chromosomes (Bs); these Bs, which are additional to the basic complement of A chromosomes, generate a spectrum of DNA amounts, and thus nucleotypes, within many species. We consider the physiological consequences of carrying extra chromosomes that appear by and large to lack coding sequences, and focus on two species with contrasting case histories: rye (Secale cereale L.) and chives (Allium schoenoprasum L.), which illustrate ‘selfish’ and ‘adaptive’ explanations of the persistence of B chromosomes.

Recent advances in the cytogenetics of the Lolium–Festuca complex provide new opportunities for understanding and manipulating complex physiological mechanisms such as drought and cold resistance. This paper describes breeding programmes involving hybrids between two species: L. multiflorum, which offers good early growth and high nutritive value, and F. arundinacea, which is more stress tolerant. The programmes are designed to allow a rapid recovery of the Lolium genome and to restrict numbers of recombinants derived from Festuca. Use of genomic in situ hybridization (GISH) and an isozyme marker demonstrates how gene complexes from any part of the Festuca genome can be introgressed into Lolium. This enables us to construct genotypes combining desirable traits of both Lolium and Festuca species. By introgressing different Festuca genes into Lolium, quantitative traits such as tolerance to drought and cold can be ‘dissected’ into their different components, to clarify their function. Festuca genes for stress tolerance can be located by genomic in situ hybridization (GISH) and assigned to regions of chromosome arms in Lolium. Two Lolium genotypes are described, in which genes for drought resistance transferred from the F. pratensis sub-genome of F. arundinacea onto chromosome 2 of Lolium. The two drought-resistant lines have the high water conductance of Festuca on their adaxial leaf surface and the low abaxial conductance of Lolium. The paper also describes how androgenesis of L. multiflorum × F. arundinacea hybrids has led to the selection and characterization of genotypes with coacclimation to drought and freezing stress, in some cases exceeding that in the Festuca parent.

Dehydrin proteins (late embryogenesis abundant (LEA) D11 family) are produced in a wide variety of plant species in response to environmental stimuli with a dehydrative component, including drought, low temperature, salinity, and developmental stages such as seed and pollen maturation. Despite their widespread occurrence and abundance in cells under dehydrative conditions, the biochemical role of dehydrins remains elusive. The subcellular location of dehydrins is consistent with a biochemical role as an intracellular stabilizer, possibly with surfactant characteristics, acting upon targets in both the nucleus and cytoplasm. In some species, dehydrin loci are located within quantitative trait loci (QTL) intervals for important phenotypic traits including winter hardiness in barley (Hordeum vulgare L.) and anthesis-silking interval in maize (Zea mays L.). Dehydrin loci tend to be multigenic and occur in clusters on more than one chromosome. Investigations are currently under way in our laboratory and others' to move beyond protein accumulation studies and correlations with QTL to uncover direct cause-and-effect relationships between dehydrin (dhn) genes and phenotypes associated with physiological responses to stress.

Salt-affected soil is a major problem and has led to large areas of the earth's surface being unable to support agriculture. Wheat is not tolerant to high levels of salt; however, a number of its wild relatives, in particular Thinopyrum bessarabicum Savul. and Rayss, which grows on the Black Sea shore in the Crimea, are tolerant to high levels of NaCl. Thus the introgression of genes conferring salt tolerance from Th. bessarabicum into Triticum aestivum is one way in which salt-tolerant wheat varieties can be produced. This paper reviews the progress made in developing salt-tolerant wheat carrying Th. bessarabicum genes and salt-tolerant wheat/Th. bessarabicum amphiploids.

Improving the drought resistance of high yielding rice (Oryza sativa L.) varieties for upland areas is a goal of rice breeders since upland rice relies exclusively on rainfall for water and is generally low yielding. Identifying quantitative trait loci (QTLs) which confer drought resistance promises to speed this goal. We have previously described the use of 82 restriction fragment length polymorphism (RFLP) markers in the genetic mapping of root growth characteristics that are potentially involved in drought resistance in a population of 178 F2 plants derived from a cross of two drought resistant upland varieties, Azucena and Bala. Here we report the characterization and QTL mapping of three other mechanisms of drought resistance, leaf rolling, stomatal behaviour and early flowering, in this population. When the youngest fully expanded leaf was excised from 12 rice varieties, ‘IR20’ and ‘Bala’ were identified as slow at rolling. Although ‘Azucena’ rolled its leaves much faster than ‘Bala’ upon excision, stomatal resistance rose more rapidly in ‘Bala’, resulting in a lower rate of water loss. By assessing leaf rolling and stomatal resistance in the F2 population, it was possible to identify QTLs for these traits. A QTL for slow leaf rolling from ‘Bala’ was found on chromosome 1. Two QTLs associated with resting stomatal conductance were found on chromosomes 3 and 12. QTLs for the rate of stomatal closure were found on chromosomes 3 and 7. In addition, it was possible to map QTLs associated with the days to heading which were found on chromosomes 3, 8 and 10. These results identify markers of potential value to breeders and are discussed in the context of previously reported data for heading date QTLs in rice and for stomatal conductance QTLs in maize.

High levels of soil aluminium place serious constraints on wheat production on acidic soils, especially in the tropical areas of Africa and South America. Conventional plant breeding has improved the tolerance of the wheat crop, but available genetic variation is limited. The wild relatives of wheat provide a valuable gene pool for the introduction of further genetic variation. One wild species, Aegilops uniaristata Vis. (2n=2x=14,NN), is being utilized as a new source of tolerance. Of the addition lines of individual N genome chromosomes of A. uniaristata to wheat (Triticum aestivum L.) which have been established and characterized, chromosome 3N has been shown to confer tolerance to wheat. The three substitution lines in which 3N replaces the homoeologous wheat chromosomes, 3A, 3B or 3D, have also been produced. Growing plants to maturity in a low pH/high Al hydroponics system confirmed that chromosome 3N conferred tolerance to the substitution lines as well as to the addition line. By manipulating the genetic control of homoeologous chromosome pairing, chromosome 3N is being recombined with its wheat homoeologues in order to introduce a smaller alien segment which carries the gene(s) for tolerance but not the agronomically unacceptable brittle rachis gene also carried on chromosome 3N.

Drought strongly affects cereal yield in several regions of the world. Plant growth and plant water status in response to soil water deficit play an important role in tolerance to drought and in yield stability. In order to investigate the relationship between plant growth and water relations, 187 barley (Hordeum vulgare L.) recombinant inbred lines from a cross between two Mediterranean varieties, Tadmor and Er/Apm, were studied in a growth chamber for RWC, number of leaves on the main tiller (NL), number of tillers (NT) and total shoot fresh and dry biomass (TSFM and TSDM). Measurements were made at the beginning of tillering on well watered plants and at a soil moisture content of 14% of field capacity. A negative phenotypic correlation between RWC and growth parameters was obtained in both treatments. Under water stress, a negative genetic correlation was also found between the same characters. QTLs involved in RWC, NL and TSFM variation were positioned on a RFLP-RAPD genetic map. Different DNA regions involved in constitutive and water stress responses were detected. In the stress treatment, one region on chromosome 1 was involved in RWC and NL variation giving a genetic basis to the phenotypic correlations found. Separated map positions were also found for RWC and NL. Epistatic interactions between several QTLs and between QTLs and other markers were detected only in the water stress treatment, suggesting that some chromosomal regions might be involved in the regulation of the expression of the traits under water stress. This work suggested that even if some parameters are strongly correlated, finding QTLs for only one trait is not sufficient to detect all the candidate regions which might be involved in the control of the correlated traits. The results of localization and co-localization of QTLs are physiologically interpreted.

Variation for K+ and Na+ accumulation at low salinities in hydroponic (water) culture were observed in shoots of different wheat species. Greater discrimination (in favour of K+ and against Na+ accumulation) was shown by hexaploid bread wheat (Triticum aestivum L.) than by tetraploid durum wheat (T. turgidum L.). Since Aegilops tauschii Cosson (A. squarrosa L.), the source of the D genome in bread wheat, also exhibited high discrimination between K+ and Na+, it was concluded that the character resided in the D genome. Studies of aneuploid bread wheat lines and disomic substitution lines of D genome chromosomes for their A and B genome homoeologues in durum wheat cv. Langdon revealed that the trait was controlled by the long arm of chromosome 4D. Since the aneuploid and disomic substitution lines showed better relative salt tolerance than durum wheat, but had lower yield potentials, we recombined chromosome 4D with chromosome 4B in a tetraploid wheat background using a homoeologous pairing mutant. This produced families of 4D/4B recombinant lines, some of which exhibited the enhanced K+/Na+ discrimination trait. RFLP analysis confirmed that the trait was controlled by a single gene (Kna1) which was completely linked to five markers on the distal third of the long arm of 4D. A second cycle of homoeologous recombination was employed to remove the distal 4D genetic material from the recombined Kna1 4B/4D chromosome and to map Kna1 in greater detail. By this strategy, Kna1 was mapped within a short 2 cM region. Genetic analysis of K+[ratio ]Na+ ratios showed very high LOD scores in this region for plants grown in solution culture, but lower values for plants grown in the field.

In general, recombinant lines which exhibited the enhanced K+/Na+ trait were slightly more tolerant of salinity in the field and in sand culture than recombinants lacking the trait. There was, however, considerable variation between individual lines. Ion discrimination and relative tolerance were also higher in a Kna1 recombinant (line no. R3) than in a kna1 recombinant (line no. R165) in sodic conditions. In these two lines the enhanced K+/Na+ discrimination trait did not alter responses to low potassium or calcium supply.

Two families of Helianthus lines, T− (low desiccation rate) and T+ (low desiccation rate), with contrasting osmotic adjustment (OA), obtained from a divergent selection programme, were compared for several morphological traits, ecophysiological parameters and molecular markers. The physiological analysis in a glasshouse experiment showed that the two families were identical in irrigated conditions except for a single trait, relative water loss (RWL: water loss rate of excised leaves). RWL could be used for these genotypes as a physiological marker of OA. A molecular analysis of the two contrasting lines, using differential screening of cDNA libraries, led to the isolation of three specific cDNAs, one of which, DRS26, showed high sequence homology with a mammalian amino-acid transporter. Subsequent RFLP and STS studies, using DRS26 and RAPD bulked analysis, enabled the identification of putative molecular markers of RWL and OA in T−[ratio ]T+ sunflower.

In this paper, the response to temperature of three physiological processes has been examined in detail in seedlings of sorghum and pearl millet. These have been compared with their field performance under high temperature conditions with the aim of developing laboratory-based screening techniques for seedling thermotolerance. Membrane thermostability, as assessed by electrolyte leakage, seedling re-growth and protein synthetic ability were measured in seedlings exposed to a range of temperatures using a thermal gradient bar. The effect of genotype, seedling age and pre-treatment was examined. Differences between genotypes were only apparent after seedlings had been given a brief high temperature pre-treatment (2 h at 43°C). Similar results were found with all three methods used and their suitability for use in the screening of large numbers of entries is discussed. Significant correlation was found between the ability of membrane thermostability to acclimate and seedling survival in the field.

This paper reviews the results of several years' study by a multidisciplinary team comprising geneticists, physiologists, ecologists and statisticians of abiotic stress tolerance in barley. The strategy involves genetic fingerprinting of wild barley lines (Hordeum spontaneum C. Koch) from a range of habitats and testing these for responses to abiotic stresses under controlled conditions. Multiple regression analysis is used to identify genetic markers associated with experimentally determined stress responses. These data are related to site-of-origin ecogeographic data and used to identify areas of useful wild variation. Markers associated with traits of interest can be mapped in genetic mapping populations, revealing areas of the barley genome carrying genes controlling stress responses. This paper highlights our work on associations of amplied fragment length polymorphisms with salt tolerance and describes some initial results of the use of SSRs in studying drought tolerance in barley.

Amplified fragment length polymorphisms (AFLPs) offer a reproducible, multiplex DNA assay by which to genotype mapping populations. We have evaluated physiological traits in barley seedlings grown in an hydroponic system and given a salt treatment. Multiple regression was used to show associations between AFLPs and quantitative traits. Effects at different loci were detected in stress treatments in comparison to the control implying that either novel gene action was induced by salt stress or that normal activity was reduced to a low level where alternate gene action is revealed. The QTLs occurred on all chromosomes but there appeared to be clusters of loci on chromosomes 1 (7H), 4 (4H), 5 (1H) and 6 (6H). The significance of our results is discussed in the context of studies to explore the barley genome and the application of the results of these genetical analyses to barley breeding.

This paper describes a developing approach for correlating molecular markers with phenotypic expression in barley, and research examples are given. This approach is intended to elucidate the connection between defined parts of the barley genome and their physiological expressions. To illustrate our approach (up to the point of marker correlation), the materials, methods and results of an experiment are presented. Briefly, the natural abundance levels of carbon and nitrogen isotopes (δ13C and δ15N) serve as the primary screening tools for assessing barley genotypes for their responses to imposed abiotic stresses. The isotopic responses are then correlated with molecular markers as the first step in identifying the mechanisms relating genetic variation with phenotypic expression.

A well-described mechanistic model exists for interpreting plant δ13C, and this enables partitioning of the physiological processes contributing to the whole plant δ13C. Although plant δ15N appears to be equally well correlated with genotype and environment, there is no mechanistic model explaining this isotopic signature. Hence, δ15N is used empirically and in parallel with attempts to understand its underlying mechanism(s).

This is a review of genetic mapping with molecular markers aimed at the non-specialist who wishes to use, or at least grasp the concepts behind, this powerful analytical tool. Restriction fragment length polymorphisms (RFLPs) are defined and used to illustrate the different aspects of mapping. The principles of segregation, recombination and linkage are considered and related to the idea of a molecular marker map. A description of a typical mapping population and how it is analysed follows. Traits to be mapped are divided into those controlled by ‘major’ genes and those governed by quantitative trait loci (QTLs). Exploitation of the map for marker-assisted selection, gene cloning and synteny comparisons is discussed, as are some of the limitations to the usefulness of molecular marker maps. Finally other marker systems are introduced, namely minisatellites or variable number tandem repeats (VNTRs); randomly amplified polymorphic DNA (RAPDs); microsatellites or simple sequence repeats (SSRs); and amplified fragment length polymorphisms (AFLPs).