Lolium perenne (4x)/Festuca pratensis (2x) triploid hybrids (2n = 3x = 21) were produced and backcrossed to Lolium perenne. The BC 1 progeny, which predominantly had 14 chromosomes, were analysed using genomic in situ hybridization (GISH) and genetic markers. GISH revealed that over 74% of the BC 1 individuals carried one or more F. pratensis chromosome segments. By comparing the physical size of introgressed F. pratensis chromosome segments with the presence or absence of F. pratensis-specific polymorphisms, it was possible to determine the physical position of genetic markers. The potential of a new type of genetic mapping ('introgression mapping') for the alignment of physical and genetic maps, determining the genetic control of agronomically important characters and the production of novel germplasm for the development of new varieties is discussed.Keywords: genetic mapping, genomic in situ hybridization, introgression mapping, physical mapping, recombination. IntroductionGenome mapping is a powerful approach to the study of plant biology, facilitating the location of genes responsible for agronomically and scientifically important traits. Phenotype mapping is the primary justification for the development of genetic maps. DNA markers closely linked to a gene responsible for a desirable phenotype are an important tool for indirect selection in breeding programmes and in addition provide a springboard from which genes can be isolated via map-based cloning. To increase the efficiency of mapping, parental genotypes which exhibit high DNA marker allele diversity are chosen. The genetic base of many crop species, however, is narrow, but this problem can be overcome by generating maps from wide crosses.The forage grass Lolium perenne (Lp) (2n = 2x = 14) can be readily hybridized with Festuca pratensis (Fp) (2n = 2x = 14) to form a 14-chromosome hybrid which exhibits full pairing at metaphase I of meiosis (Lewis, 1966;Jauhar, 1975). These diploid hybrids show nearly complete sterility (Lewis, 1966;Jauhar, 1975). However, LpLpFp triploids, derived by hybridizing synthetic tetraploid L. perenne with diploid F. pratensis, show both male and female fertility (Jauhar, 1975). The ease of hybridization, the chromosome pairing observed at meiosis and a preliminary molecular genetic study on the phylogeny of these species (Stammers et al., 1995) indicate that they are closely related. In contrast, the sterility of the diploid hybrid, the ease with which Lp and Fp chromosomes can be distinguished using genomic in situ hybridization (GISH) , and the position and number of rDNA sites in the two species (Thomas et al., 1996; indicates that they are distantly related. Thus the phylogenetic relationship between Lp and Fp at present remains a paradox.The work presented in this paper describes a new kind of genetic mapping in the grasses. 'Introgression mapping' is based on the ability to distinguish *Correspondence. E-mail: ian.king@bbsrc.ac.uk ©1998 The Genetical Society of Great Britain. 462Lp and Fp chromosomes and chromosome se...

Lolium perenne (4x)/Festuca pratensis (2x) triploid hybrids (2n = 3x = 21) were produced and backcrossed to Lolium perenne. The BC 1 progeny, which predominantly had 14 chromosomes, were analysed using genomic in situ hybridization (GISH) and genetic markers. GISH revealed that over 74% of the BC 1 individuals carried one or more F. pratensis chromosome segments. By comparing the physical size of introgressed F. pratensis chromosome segments with the presence or absence of F. pratensis-specific polymorphisms, it was possible to determine the physical position of genetic markers. The potential of a new type of genetic mapping ('introgression mapping') for the alignment of physical and genetic maps, determining the genetic control of agronomically important characters and the production of novel germplasm for the development of new varieties is discussed.Keywords: genetic mapping, genomic in situ hybridization, introgression mapping, physical mapping, recombination. IntroductionGenome mapping is a powerful approach to the study of plant biology, facilitating the location of genes responsible for agronomically and scientifically important traits. Phenotype mapping is the primary justification for the development of genetic maps. DNA markers closely linked to a gene responsible for a desirable phenotype are an important tool for indirect selection in breeding programmes and in addition provide a springboard from which genes can be isolated via map-based cloning. To increase the efficiency of mapping, parental genotypes which exhibit high DNA marker allele diversity are chosen. The genetic base of many crop species, however, is narrow, but this problem can be overcome by generating maps from wide crosses.The forage grass Lolium perenne (Lp) (2n = 2x = 14) can be readily hybridized with Festuca pratensis (Fp) (2n = 2x = 14) to form a 14-chromosome hybrid which exhibits full pairing at metaphase I of meiosis (Lewis, 1966;Jauhar, 1975). These diploid hybrids show nearly complete sterility (Lewis, 1966;Jauhar, 1975). However, LpLpFp triploids, derived by hybridizing synthetic tetraploid L. perenne with diploid F. pratensis, show both male and female fertility (Jauhar, 1975). The ease of hybridization, the chromosome pairing observed at meiosis and a preliminary molecular genetic study on the phylogeny of these species (Stammers et al., 1995) indicate that they are closely related. In contrast, the sterility of the diploid hybrid, the ease with which Lp and Fp chromosomes can be distinguished using genomic in situ hybridization (GISH) , and the position and number of rDNA sites in the two species (Thomas et al., 1996; indicates that they are distantly related. Thus the phylogenetic relationship between Lp and Fp at present remains a paradox.The work presented in this paper describes a new kind of genetic mapping in the grasses. 'Introgression mapping' is based on the ability to distinguish *Correspondence. E-mail: ian.king@bbsrc.ac.uk ©1998 The Genetical Society of Great Britain. 462Lp and Fp chromosomes and chromosome se...

Programmed plant cell death is a widespread phenomenon resulting in the formation of xylem vessels, dissected leaf forms, and aerenchyma. We demonstrate here that some characteristics of programmed cell death can also be observed during the cellular response to biotic and abiotic stress when plant tissue is ingested by grazing ruminants. Furthermore, the onset and progression of plant cell death processes may influence the proteolytic rate in the rumen. This is important because rapid proteolysis of plant proteins in ruminants is a major cause of the inefficient conversion of plant to animal protein resulting in the release of environmental N pollutants. Although rumen proteolysis is widely believed to be mediated by proteases from rumen microorganisms, proteolysis and cell death occurred concurrently in clover leaves incubated in vitro under rumenlike conditions (maintained anaerobically at 39 degrees C) but in the absence of a rumen microbial population. Under rumenlike conditions, both red and white clover cells showed progressive loss of DNA, but this was only associated with fragmentation in white clover. Cell death was indicated by increased ionic leakage and the appearance of terminal deoxynucleotidyl transferase-mediated dUTP-nick-end-labelled nuclei. Foliar protein decreased to 50% of the initial values after 3 h incubation in white clover and after 4 h in red clover, while no decrease was observed in ambient (25 degrees C, aerobic) incubations. In white clover, decreased foliar protein coincided with an increased number of protease isoforms.

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.

Production of nitrogenous waste by livestock agriculture is a significant environmental concern in terms of pollution of land and water. In the rumens of cattle and sheep, the excessive proteolysis which contributes to inefficiency of nutrient use involves both the rumen microbial population and the intrinsic plant proteases that can mediate protein degradation in ingested fresh forage on exposure to the environmental stresses of the rumen. Here, white clover (Trifolium repens) plants that do not form root nodules, and so are dependent on nitrate supplied to the roots, have been used to determine how nitrogen status of the plant affects the rate of plant‐mediated proteolysis in forage under conditions that simulate ingestion by grazing ruminants. Plants were grown from seed and supplied with nutrient solution containing 2.5, 5.0, 7.5 or 10 mM nitrate. Protein, free amino acid and protease activity were determined in leaves which had been placed in an in vitro system designed to simulate conditions experienced in the rumen (anaerobic phosphate buffer maintained at 39°C in the dark). Foliar protein content increased with increasing nitrate supply, while in vitro incubation of leaves resulted in time‐dependent decreases in protein concentration and increases in amino acid concentration. Regardless of nitrate supply, 50% of the protein was degraded in 6 h and 80% after 24 h. As the extent of protein decrease was determined by initial protein content, more protein degradation occurred in those plants grown with the highest nitrate supply: after 6 h, 130.7 mg g−1 dry matter (DM) was degraded in leaves grown at 10 mM nitrate but only 52.3 mg g−1 DM in leaves grown at 2.5 mM nitrate. Hence, although the percentage of proteolysis is independent of foliar protein concentration, the latter is critical to the quantity of protein degraded. Heat‐stable serine and cysteine proteases were active throughout the term of the in vitro incubation. Although proteolysis in ingested forage can continue for many hours, mediated by heat‐stable proteases, maximum amino acid accumulation accounted for less than 40% of initial protein. Therefore, it is proposed that continued and extensive proteolysis occurs following leaf excision and exposure to rumen conditions because amino acid accumulation is insufficient to initiate those feedback systems which sense cytoplasmic amino acid concentration and prevent excessive proteolysis during normal source–sink relations.