The effect of combination treatment with trenbolone acetate and estradiol-17ß on skeletal muscle expression and plasma concentrations of oxytocin in sheep.

Implantation of trenbolone acetate (TBA) in conjunction with estradiol-17ß (E(2)) increases growth, feed conversion efficiency, and carcass leanness in cattle. Our previous study in Brahman steers suggested that the neuropeptide hormone oxytocin (OXT) may be involved in increasing muscle growth after TBA-E(2) treatment. The present study aimed to determine whether OXT mRNA expression in the longissimus muscle (LM) is also up-regulated in TBA-E(2-)implanted wethers as has been found in steers. Real-time quantitative PCR was used to measure the expression of the gene encoding the OXT precursor, three genes with increased expression in the LM muscle of TBA-E(2)-treated steers, MYOD1 (muscle transcription factor), GREB1 (growth regulation by estrogen in breast cancer 1), and WISP2 (Wnt-1 inducible signaling pathway protein 2), and two genes encoding IGF pathway proteins, IGF1, IGFR, in the LM of both untreated and TBA-E(2)-treated wethers. The expression of OXT mRNA in wethers that received the TBA-E(2) treatment was increased ~4.4-fold (P = 0.01). TBA-E(2) treatment also induced a 2.3-fold increase in circulating OXT (P = 0.001). These data, together with the observation that untreated wethers had much higher baseline concentrations of circulating OXT than previously observed in steers, suggest that wethers and steers have quite different OXT hormone systems. TBA-E(2) treatment had no effect on the expression of IGF1, IGFR, and the muscle regulatory gene MYOD1 mRNA levels in wethers (P ≥ 0.15), but there was an increase in the expression of the two growth-related genes, GREB1 (P = 0.001) and WISP2 (P = 0.04). Both genes are common gene targets for both the estrogen and androgen signaling pathways. Consequently, their actions may contribute to the positive interaction between TBA and E(2) on additive improvements on muscle growth.

Genetic diversity in European Pisum germplasm collections.

The distinctness of, and overlap between, pea genotypes held in several Pisum germplasm collections has been used to determine their relatedness and to test previous ideas about the genetic diversity of Pisum. Our characterisation of genetic diversity among 4,538 Pisum accessions held in 7 European Genebanks has identified sources of novel genetic variation, and both reinforces and refines previous interpretations of the overall structure of genetic diversity in Pisum. Molecular marker analysis was based upon the presence/absence of polymorphism of retrotransposon insertions scored by a high-throughput microarray and SSAP approaches. We conclude that the diversity of Pisum constitutes a broad continuum, with graded differentiation into sub-populations which display various degrees of distinctness. The most distinct genetic groups correspond to the named taxa while the cultivars and landraces of Pisum sativum can be divided into two broad types, one of which is strongly enriched for modern cultivars. The addition of germplasm sets from six European Genebanks, chosen to represent high diversity, to a single collection previously studied with these markers resulted in modest additions to the overall diversity observed, suggesting that the great majority of the total genetic diversity collected for the Pisum genus has now been described. Two interesting sources of novel genetic variation have been identified. Finally, we have proposed reference sets of core accessions with a range of sample sizes to represent Pisum diversity for the future study and exploitation by researchers and breeders.

Anthelmintic efficacy of five tropical native Australian plants against Haemonchus contortus and Trichostrongylus colubriformis in experimentally infected goats (Capra hircus).

The study of the anthelmintic properties of plants rich in plant secondary metabolites can provide ecologically sound methods for the treatment of parasites on grazing animals. The purpose of the present study was to evaluate the anthelmintic effect of five tropical native Australian plant species rich in plant secondary metabolites on adult Haemonchus contortus and Trichostrongylus colubriformis in experimentally infected goats. Thirty young, nematode-free goats were infected with 2500 H. contortus and 5000 T. colubriformis infective larvae thrice weekly for a week (day 1-7 of the experiment). On day 27 after first infection, the goats were allocated into six groups of five animals per group. From day 28 to day 35, fresh leaves from Acacia salicina, Acacia nilotica, Eucalyptus corymbia, Casuarina cunninghamiana and Eucalyptus drepanophylla were included in the goats diet. Five groups were offered leaves from one of these plant species and one group, the untreated control, received only the basal diet formulated with 20% Medicago sativa and 80% Avena sativa. Following plant material administration, the goats were monitored daily until day 40 and then slaughtered on day 41. Total faecal worm egg output, total production of larvae recovered from faecal cultures, total post-mortem worm burdens and the per capita fecundity of female worms were estimated. The toxicity of the plant species for the goats was measured by histopathological analyses of liver and kidney samples. Results showed that goats feeding on the plant material rich in plant secondary metabolites had significantly lower egg output compared to the control goats (P<0.05). A similar response was found for larval production in both H. contortus and T. colubriformis supporting that egg output was affected in both species. Although the total worm burdens were not affected by the plant material (P>0.05), the per capita fecundity was significantly reduced by E. corymbia, A. nilotica and A. salicina (P<0.05). No signs of toxicity were detected in the liver or kidney samples. It is concluded that goats can benefit from the short-term ingestion of plant secondary metabolites, which reduce the total faecal egg output and thus decrease the potential for re-infection from the pasture.

Novel approaches for the control of helminth parasites of livestock VI: summary of discussions and conclusions.

Gastrointestinal helminth parasites impact on livestock production systems throughout the world, and the use of anthelmintics to control this problem has lead to the inevitable development of populations of helminths resistant to these treatments. This, coupled with consumer desires for minimal chemical inputs into food and fibre production, has prompted research into non-chemical approaches to helminth control. Scientists of the "Novel Approaches to the Control of Helminth Parasites of Livestock" group met for the 6th time in August 2010 and this paper summarises that meeting. Six scientific sessions addressed current approaches and topics of interest through formal presentations and discussion of issues raised by the contributing authors. Close interaction between researchers and extension specialists during the meeting has contributed to enhanced prospects for field application of research outcomes in the future.

Role of host and environment in mediating reduced gastrointestinal nematode infections in sheep due to intensive rotational grazing.

We have previously reported marked reductions in faecal worm egg counts (WECs) and drenching frequency in sheep on an intensive rotational grazing system (IRG) in a cool temperate environment with summer-dominant rainfall. These experiments were designed to determine the role of the host and environmental factors in mediating this. The role of host factors was investigated by administering a fixed larval challenge in each of the 4 seasons of the year to groups of 20 young sheep on three different management systems, including IRG. This comprised a mixed larval challenge containing infective larvae of Haemonchus contortus and Trichostrongylus colubriformis 7 days after short-acting anthelmintic treatment. A range of measurements was then made up to day 35 post-challenge. The role of environmental factors was determined by assessing pasture infectivity in four seasons using faecal worm egg counts (WECs) and pooled faecal culture of worm-free tracer sheep. The management systems were high input (HI) with high fertiliser inputs high stocking rate and relatively long grazing periods; typical New England management system (TYP) with moderate fertiliser inputs and stocking rate and relatively long grazing periods and; Intensive rotational grazing (IRG) with moderate fertiliser inputs and stocking rate but very short (mean 5 days) grazing periods and long (mean 103 days) rest periods. IRG sheep had higher mean WEC at 28 and 35 days after fixed larval challenge than HI and TYP sheep in spring (IRG: 9500 ± 1000; HI: 4000 ± 1000; TYP: 7200 ± 1000 eggs/g, P<0.01) and summer (IRG: 8400 ± 750; HI: 5300 ± 800; TYP: 4400 ± 700 eggs/g; P<0.001) and also had lower live weights during these seasons. There was no difference in WEC after the autumn challenge (IRG: 5100 ± 450 HI: 4500 ± 450; TYP: 4200 ± 450 eggs/g; P ≈ 0.36) but IRG had lower WEC than TYP following the winter challenge (IRG: 2900 ± 400; HI: 2300 ± 400; TYP: 4300 ± 400 eggs/g, P<0.01). The tracer sheep (used to determine pasture infectivity) on IRG had significantly lower WECs during winter, spring and summer than those under the other management systems. Faecal culture and larval differentiation revealed that faeces from tracers on IRG contained significantly lower proportions of H. contortus and significantly higher proportions of Trichostrongylus spp. and Teladorsagia circumcincta than faeces from tracers on the HI and TYP treatments. Thus, when IRG was most efficacious for worm control, during spring and summer when short graze and long rest periods were maintained, sheep on this system exhibited greater susceptibility to larval challenge while tracer sheep indicated lower pasture infectivity. This demonstrates that the effects of IRG on WEC are mediated by reduced larval challenge rather than increased host resistance to infection.

The use of DNA markers to map anthelmintic resistance loci in an intraspecific cross of Haemonchus contortus.

The use of DNA markers to track the development of anthelmintic resistance in parasites of livestock would allow informed choices for the management of this important problem. We describe a genetic mapping approach for the discovery of DNA markers for anthelmintic resistance in Haemonchus contortus. We crossed a multi-drug resistant field isolate of H. contortus with a well-characterized laboratory strain susceptible to 4 drug classes. The F2 were separately selected with 5 anthelmintics from 4 drug classes, producing drug-resistant populations carrying gene variants derived from both the field isolate and the laboratory strain. Individual F2 worms were analysed using amplicon length polymorphisms (ALPs). We looked for field isolate alleles over- or under-represented in F2 populations compared to the unselected F2 and/or the laboratory strain. The data we obtained suggest that marker association can be used to link neutral markers with resistance, but also that more markers and perhaps more inbred laboratory strains would make the procedure more likely to succeed.

Helminth control using local resources in smallholder production systems of Asia.

Practical helminth control in smallholder systems needs to match technical options with local knowledge, locally available animal and feed resources and the needs of both producers and consumers. Despite extensive research over many decades the uptake of new technology in these systems has been slow and limited to few farmers with access to good technical support. Investment by small holders is constrained by lack of regular market signals for livestock and livestock products. Examples of effective helminth control in cattle, buffalo, sheep, goat and pig systems show that effective control is possible using local resources and knowledge. Livestock systems in Asia are changing rapidly through industrialisation of production and consolidation of supply chains and retail markets, but smallholder systems, which are resilient and multipurpose, will remain important in rural areas and in peri-urban environments. They also provide a significant pathway for the poor to build assets and generate income. Helminth control in these systems will always vary greatly between farmers and systems and will need to be relatively simple and tailored to locally available resources. The public sector will continue to provide advice on appropriate genetics, to regulate drug importation, use and quality, and ensure novel helminth control options are investigated for local application and promotion to livestock producers. The private sector has the complementary role to develop clear market signals for livestock and livestock products, and make anthelmintics available in appropriate packages. Improved helminth control has the potential to increase the profitability and sustainability of all components of the livestock sector.

Intensive rotational grazing assists control of gastrointestinal nematodosis of sheep in a cool temperate environment with summer-dominant rainfall.

While rotational grazing methods have an accepted role in the management of gastrointestinal nematodosis (GIN) of small ruminants in humid tropical regions, their efficacy and application in cool temperate regions is more controversial. This study evaluated GIN over 2 years in three classes of fine wool Merino sheep (lambs, hoggets and ewes) under three different sheep management systems in a cool tableland environment near Armidale NSW Australia (950m altitude, 30 degrees 31'S, 151 degrees 39'E). The management systems were High input (HI) with high fertiliser inputs, a target of 100% sown pasture, high stocking rate (13.4 dry sheep equivalents/ha) and relatively long grazing periods; Typical (TYP) New England management system with moderate fertiliser inputs and stocking rate (9.3DSE/ha) and relatively long grazing periods; and Intensive rotational grazing (IRG) with moderate fertiliser inputs and stocking rate (8.8DSE/ha) but very short (mean 5 days) grazing periods and long (mean 103 days) rest periods. Twenty sheep of each class in each management treatment were sampled monthly for faecal worm egg counts (WEC, followed by larval differentiation), and body weights with a blood sample taken for haematology every second month. The proportion of sheep with WEC above zero did not differ between management systems but the magnitude of WEC did, with sheep under IRG displaying lower mean WEC than those on the other treatments (IRG: 326, HI: 594, TYP: 536, eggs/g P<0.0001). This was despite a significantly longer mean interval between anthelmintic treatments (IRG: 144 days, HI: 77 days, TYP: 78 days, P<0.0001). The IRG management system also influenced the composition of the infections with sheep on this treatment having a significantly lower proportion of Haemonchus contortus in their faecal cultures (IRG: 59.7%, HI: 79.4%, TYP: 80.9%, P<0.05) and a significantly higher proportion of Trichostrongylus spp. Sheep on the IRG treatment also had a significantly higher haematocrit (HCT) than those on the other management systems. Despite the lower WEC and higher HCT, sheep under IRG also had significantly lower bodyweights and fleece weights overall, although this was only evident in sheep raised to adulthood prior to the experiment, not those raised during the 2-year experimental period. The results demonstrate that IRG systems with short grazing periods and long rest periods between grazing events can assist with control of GIN in cool temperate climates where H. contortus is the dominant parasite.

Genetic and phenotypic differences between isolates of Haemonchus contortus in Australia.

We believe this study is the first to consider the genetic and phenotypic divergence between isolates of Haemonchus contortus in Australia. Microsatellite markers have been used to investigate genetic divergence, whilst phenotypic divergence has been considered through individual worm morphology, isolate life history traits and the effect of isolates upon the host. The results are discussed in the context of the likely introduction of H. contortus to Australia, its recent isolation, and the characteristics of sheep and goat farming which might act to either isolate or distribute parasites. We conclude that there is significant observable genetic divergence between isolates of H. contortus in Australia. The divergence may have been under-estimated in this study due to a variety of factors. Phenotypic divergence is also observed, and potentially has significant implications for both economic losses due to haemonchosis on individual properties and for decisions regarding the regulation of stock movements in Australia.

Exploiting the effect of dietary supplementation of small ruminants on resilience and resistance against gastrointestinal nematodes.

This paper focuses on targeted nutritional supplementation as a means to reduce the requirement for chemotherapeutic control of gastrointestinal nematode infection of small ruminants and considers the limitations to practical application. Supplementary feeding, particularly with additional dietary protein, can assist resilience to infection during times when metabolic resources are being directed towards dealing with the pathophysiological effects of infection and away from production of meat, milk and fibre. Substantial experimental evidence from studies of both sheep and goats supports this hypothesis particularly in relation to young lambs and kids after weaning and in ewes around parturition. In addition, nutritional supplementation frequently increases resistance to infection, as indicated by decreased faecal worm egg counts and worm burdens. As a result, supplementation has the potential to reduce the requirement for anthelmintic treatment. Practical application of this knowledge can, however, be quite complex in many small ruminant production systems. In general, strategic supplementation should target those times when nutrient requirements are greatest and provide those nutrients which are deficient whether protein, energy, minerals or trace elements. Complexity arises when we consider that nutrient requirements will differ between localities for different species and breed of host, at different stages of growth and reproduction, with differing seasonal availability of forage, with different species of nematodes and different levels of established infections and exposure to infective stages. As a starting point, the provision of nutrients to optimize rumen function and animal performance in the particular production system should assist in maintaining resilience to nematode infection. Provision of nutrients in excess of this requirement, if economically feasible, may yield further benefits in some situations and reduce the need for alternative control measures for gastrointestinal nematode parasites.

Novel approaches to control of parasites­a workshop.

With the advent of helminth parasite populations that have developed resistance to anthelmintics over the last decade or so, especially in small ruminants, sustainable productivity has been threatened. This workshop on novel approaches to control was held at the 19th International Conference of the World Association for the Advancement of Veterinary Parasitology (WAAVP) at New Orleans,LA, USA, during 10­14 August 2003. The workshop was organized and chaired by J.E. Miller and P.J. Waller. Novel or alternative approaches to control have been the focus of research (basic and applied) in many parts of the world. The objective of the workshop was to discuss where we have been and what direction(s) appears to be viable for both the short and long term future. In the long term, all represented regions at the workshop have promulgated programs where breeding for resistance may be the best approach as genes for resistance can be fixed in host populations. However,it does take many years to achieve results and the question of tradeoff concerning alteration of production traits needs further evaluation. Vaccination, especially against Haemonchus contortus,has been a thrust of laboratories in Scotland and Australia where natural "hidden gut" antigens have shown promise, but recombinant products have yet to be developed. In Europe, North and South America, Australia, South Africa and Asia, biocontrol using the nematode-trapping fungus Duddingtonia flagrans has been shown to be effective under experimental conditions, but some field evaluations have been disappointing. Most recently, the FAMACHA system was developed in South Africa. This system is directly and immediately applicable to all regions where H. contortus is a problem. Although not a new or novel approach, copper-oxide wire particles have been revived as a means to control H. contortus.Work being done in Europe, North and South America,South Africa and Australia have shown very encouraging results and can probably be considered the best short term approach available. However, caution needs to be considered in sheep to avoid potential copper toxicity problems. Work in New Zealand, Scotland and the US with forages and feeds containing condensed tannins have shown some limited control. Many laboratories have demonstrated that adequate and balanced nutrition programs are also important to maintain mechanisms that combat infections. Overall, no one approach alone is the answer. Approaches that are integrated, including "smart" use of anthelmintics, are necessary.

Regulation of the resistance to nematode parasites of single- and twin-bearing Merino ewes through nutrition and genetic selection.

Periparturient Merino ewes obtained from lines of sheep that had been selected either for increased resistance to Haemonchus contortus (R) or at random (C) were supplemented, while grazing at pasture, with either nil or 250 g/day cottonseed meal (CSM) for the 6 weeks prior to or the 6 weeks after the start of parturition. Ewes from both supplement groups had lower (mean 66% reduction) faecal egg counts (FECs) during the postpartum period and this coincided with a period of maternal body weight loss. Factors which increased the rate of maternal body weight loss, such as pregnancy and lactation status, also increased FEC. Evidence is presented that the magnitude of the periparturient rise (PPR) in FEC in grazing ewes will be greatest during periods of maternal weight loss and at these times supplementation to increase metabolisable protein (MP) supply will be most effective in increasing resistance to nematode parasites. The resistance of R ewes to nematode parasites was greater than that of C ewes throughout the experiment and was sufficiently low such that anthelmintic treatment in a commercial environment may not have been required. Irrespective of actual FEC, ewes from all treatment combinations exhibited a PPR in FEC. Reduced FEC of R ewes resulted in reduced apparent pasture larval contamination after 18 weeks of continuous grazing but supplementation was ineffective in this regard. It is suggested that integrated parasite management (IPM) programs for periparturient ewes should make use of both protein supplementation and genetic selection to increase worm resistance and reduce dependency on anthelmintics for worm control.

Enhancing immunity to nematode parasites in single-bearing Merino ewes through nutrition and genetic selection.

The effectiveness of protein supplementation and genetic selection to enhance the resistance of periparturient Merino ewes to infection from gastrointestinal parasites was tested in a replicated grazing experiment. One hundred and twenty ewes from lines selected for increased resistance (R) to Haemonchus contortus or at random (C) were subjected to one of the three supplement groups that provided 0 or 250 g per day cottonseed meal for 5 weeks prior to, or for 6 weeks after the start of lambing. Faecal egg counts (FEC) of R ewes were consistently lower than those of C ewes but both groups exhibited a periparturient rise in FEC. Supplementation during the pre-partum period reduced FEC and increased ewe body weight gain. The benefits of pre-partum supplementation in reducing FEC continued to be apparent up to 10 weeks after supplementation ceased. There was a strong suggestion that the benefits to parasite resistance from protein supplementation were greatest in C ewes. Wool growth rates (15%) and birth weights (5%) were greater for C ewes but differences between the lines for lamb body weight had disappeared by day 97. The greatest benefit to resistance from protein supplementation was observed when ewes were experiencing a loss of maternal body weight. Conversely, no benefits to resistance were observed when ewes had moderate (78-107 g per day) rates of maternal weight gain. These results suggest that increased resistance as a result of protein supplementation is dependent on the prevailing supply and demand for scarce nutrients such as metabolisable protein (MP). Both genetic selection and protein nutrition are effective strategies to enhance host resistance to nematode infection during the periparturient period.

Excess heterozygosity contributes to genetic map expansion in pea recombinant inbred populations.

Several plant genetic maps presented in the literature are longer than expected from cytogenetic data. Here we compare F(2) and RI maps derived from a cross between the same two parental lines and show that excess heterozygosity contributes to map inflation. These maps have been constructed using a common set of dominant markers. Although not generally regarded as informative for F(2) mapping, these allowed rapid map construction, and the resulting data analysis has provided information not otherwise obvious when examining a population from only one generation. Segregation distortion, a common feature of most populations and marker systems, found in the F(2) but not the RI, has identified excess heterozygosity. A few markers with a deficiency of heterozygotes were found to map to linkage group V (chromosome 3), which is known to form rod bivalents in this cross. Although the final map length was longer for the F(2) population, the mapped order of markers was generally the same in the F(2) and RI maps. The data presented in this analysis reconcile much of the inconsistency between map length estimates from chiasma counts and genetic data.

Effectiveness of copper oxide wire particles for Haemonchus contortus control in sheep.

OBJECTIVE: To assess the efficacy of copper oxide wire particles (COWP) for the control of H contortus infections in grazing sheep. PROCEDURE: In experiment 1, 40 worm-free Merino hoggets (11 to 12 months of age) were divided into four equal groups and allocated to separate 0.8 ha pasture plots. Two groups then received 2.5 g COWP whereas the other two groups were untreated. From 1 week after COWP treatment all lambs received a weekly infection of 2000 H contortus larvae. At week 8, six sheep from the untreated group were then allocated to two groups and treated with either 2.5 or 5.0 g of COWP to establish therapeutic efficacy of treatment. Experiment 2 followed a similar protocol but was conducted with 40 worm-free Merino lambs (3 to 4 months of age) and no assessment of therapeutic efficacy was made. RESULTS: In experiment 1 no significant difference in faecal worm egg counts was observed between treatments and faecal worm egg counts remained less than 3000 epg in all animals. Total worm counts were reduced by 37% by COWP treatment (P = 0.055). Both 2.5 g and 5.0 g doses of COWP at 8 weeks of infection reduced faecal worm egg counts by > 85% with the higher dose giving an earlier response to treatment. In experiment 2, faecal worm egg counts at 4 and 6 weeks were reduced by more than 90% in the COWP treated lambs and worm numbers were 54% lower after 6 weeks when all remaining untreated lambs had to be treated for haemonchosis. Mean faecal worm egg counts in the COWP lambs remained below 3500 epg and clinical disease did not develop in the majority of lambs before the end of the experiment at 10 weeks. CONCLUSION: Treatment with COWPs appears to have the potential to reduce establishment and worm fecundity of Haemonchus spp for an extended period and may offer livestock producers a supplementary means of reducing larval contamination of pasture particularly in areas where anthelmintic resistance is a problem and copper supplementation is likely to be beneficial.

The potential of nematophagous fungi to control the free-living stages of nematode parasites of sheep: feeding and block studies with Duddingtonia flagrans.

A series of feeding trials was conducted with penned sheep harboring Trichostrongylus colubriformis infections. They were offered barley grains supporting the growth of the nematophagous fungus Duddingtonia flagrans. It was shown that as little as 5g of grain/sheep per day was sufficient to virtually eliminate larval numbers from faecal culture. This effect persisted for the time that the fungal grains were fed, and for up to 2 days following cessation of feeding this material. Macerated fungal grains were also incorporated into a range of feed block formulations. In all these, D. flagrans was found to survive the manufacturing process and resulted in significant reductions in larval numbers in faecal cultures set up during the feeding period to sheep. This was observed even for sheep that showed only modest and irregular block consumption. These studies demonstrate that supplementary feeding or block administration offer potential deployment options for D. flagrans as a means of biological control of nematode parasites of livestock.

Biological control of field infections of nematode parasites of young sheep with Duddingtonia flagrans and effects of spore intake on efficacy.

A field study was undertaken to determine the effects of feeding Duddingtonia flagrans to young Merino sheep on pasture. A total of 60 mixed sex lambs 4-5 months old were divided into six even groups on the basis of liveweight. On Monday to Friday, each week for 6 months, three groups were offered barley grains on which D. flagrans had been cultured while the other three groups remained untreated. Every 4 weeks liveweights were recorded and faecal samples collected for nematode egg count estimation. Feeding D. flagrans reduced faecal egg counts and tended to improve liveweight gains, but considerable differences were observed between groups within treatment. These differences are thought to result from variations between the groups in consumption of the treated barley with the "best" consumers showing the greater effects of treatment.

Stability and inheritance of methylation states at PstI sites in Pisum.

The nuclear genome of pea is heavily methylated and the stability of this methylation pattern is unknown. In this study we investigated the stability of DNA methylation and its contribution to restriction fragment polymorphism as judged by AFLP fragment differences. To do this the results of a conventional AFLP analysis were compared with those obtained by a related procedure performed on pre-amplified DNA which provided a 5-methylcytosine-free DNA template (SDAFLP). Genetic mapping in a recombinant inbred (RI) population showed that polymorphisms attributable to different methylation states of PstI sites were abundant, and generally appeared to be stably inherited, although occasional failures of the inheritance of methylation states have been found. Assessments of genetic diversity by AFLP and SDAFLP were in general agreement with each other and with the currently accepted phylogeny of Pisum, but within cultivated groups the number of differences appeared to be exaggerated by AFLP. The data suggest that epigenetic differences may have played a role in the domestication of pea.

Nodule-expressed Cyp15a cysteine protease genes map to syntenic genome regions in Pisum and Medicago spp.

PsCyp15a is a gene that encodes a vacuolar cysteine protease expressed in wilt-induced shoots of Pisum sativum (pea) and in root nodules. To further the understanding of nodular PsCyp15a expression, a region 5' to the coding sequence of the gene was cloned. Varying lengths of 5' untranslated sequence were fused with the uidA coding region and introduced from Agrobacterium rhizogenes into "hairy roots" of Vicia hirsuta. In this transgenic root nodulation assay, a promoter sequence of 900 bp was sufficient to give an expression pattern indistinguishable from that obtained in pea nodules by in situ hybridization. An orthologue of PsCyp15a was cloned from nodule mRNA of Medicago sativa and a corresponding gene identified in M. truncatula was also shown to express strongly in nodules. With molecular mapping techniques, it was demonstrated that these genes map to a syntenic genome location in pea and Medicago spp., but the map positions of the Cyp15a genes cannot be correlated with existing nodulation mutants.