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Sequence-based markers have added a new dimension in the efficiency of identifying alien introgressions in wheat. Expressed sequence tag-sequence tagged sites (EST-STS) markers have proved useful in tracing alien chromatin. In this study, we report the development of Thinopyrum bessarabicum- and Secale anatolicum-specific EST-STS markers and their application in tracing respective alien chromatin introgressions in wheat. The parental lines, Chinese Spring (CS), ISR991.1 (CS/Th. bessarabicum amphidiploid), and ISR1049.2 (CS/Secale anatolicum amphidiploid), were used as core experimental materials. Using comparative analysis of RNA-Seq data, 10 903 and 10 660 candidate sequences specific to Th. bessarabicum and S. anatolicum, respectively, were assembled and identified. To validate the genome specificity of these candidate sequences, 68 and 64 EST-STS markers were developed from randomly selected candidate sequences of Th. bessarabicum and S. anatolicum, respectively, and tested on sets of alien addition lines. Fifty-five and 53 markers for Th. bessarabicum and S. anatolicum chromatin, respectively, were assigned to chromosomal location(s), covering all seven chromosomes. Approximately 83% of S. anatolicum-specific markers were transferable to S. cereale. The genome-specific candidate sequences identified and the EST-STS markers developed will be valuable resources for exploitation of Th. bessarabicum and Secale species diversity in wheat and triticale breeding.

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Emmer wheat (Triticum dicoccon Schrank) is a potential source of new genetic diversity for the improvement of hexaploid bread wheat. Emmer wheat was crossed and backcrossed to bread wheat and 480 doubled haploids (DHs) were produced from BC1F1 plants with hexaploid appearance derived from 19 crossses. These DHs were screened under well-watered conditions (E1) in 2013 to identify high-yielding materials with similar phenology. One-hundred and eighty seven DH lines selected on this basis, 4 commercial bread wheat cultivars and 9 bread wheat parents were then evaluated in extensive field experiments under two contrasting moisture regimes in north-western NSW in 2014 and 2015. A significant range in the water-use-efficiency of grain production (WUEGrain) was observed among the emmer derivatives. Of these, 8 hexaploid lines developed from 8 different emmer wheat parents had significantly improved intrinsic water-use-efficiency (WUEintr) and instantaneous water-use-efficiency (WUEi) compared to their bread wheat recurrent parents. Accurate and large scale field-based phenotyping was effective in identifying emmer wheat derived lines with superior performance to their hexaploid bread wheat recurrent parents under moisture stress.

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Potato is the 4th largest staple food in the world currently. As a high biomass crop, potato harbors excellent potential to produce energy-rich compounds such as triacylglycerol as a valuable co-product. We have previously reported that transgenic potato tubers overexpressing WRINKLED1, DIACYLGLYCEROL ACYLTRANSFERASE 1, and OLEOSIN genes produced considerable levels of triacylglycerol. In this study, the same genetic engineering strategy was employed on potato leaves. The overexpression of Arabidopsis thaliana WRINKED1 under the transcriptional control of a senescence-inducible promoter together with Arabidopsis thaliana DIACYLGLYCEROL ACYLTRANSFERASE 1 and Sesamum indicum OLEOSIN driven by the Cauliflower Mosaic Virus 35S promoter and small subunit of Rubisco promoter respectively, resulted in an approximately 30- fold enhancement of triacylglycerols in the senescent transgenic potato leaves compared to the wild type. The increase of triacylglycerol in the transgenic potato leaves was accompanied by perturbations of carbohydrate accumulation, apparent in a reduction in starch content and increased total soluble sugars, as well as changes of polar membrane lipids at different developmental stages. Microscopic and biochemical analysis further indicated that triacylglycerols and lipid droplets could not be produced in chloroplasts, despite the increase and enlargement of plastoglobuli at the senescent stage. Possibly enhanced accumulation of fatty acid phytyl esters in the plastoglobuli were reflected in transgenic potato leaves relative to wild type. It is likely that the plastoglobuli may have hijacked some of the carbon as the result of WRINKED1 expression, which could be a potential factor restricting the effective accumulation of triacylglycerols in potato leaves. Increased lipid production was also observed in potato tubers, which may have affected the tuberization to a certain extent. The expression of transgenes in potato leaf not only altered the carbon partitioning in the photosynthetic source tissue, but also the underground sink organs which highly relies on the leaves in development and energy deposition.

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KEY MESSAGE: A variety of Thinopyrum bessarabicum introgressions in both hexaploid and tetraploid wheats were generated and characterized by molecular cytogenetic analysis. Six wheat-J genome recombinants were identified with ND-FISH and GISH. Diploid wheatgrass, Thinopyrum bessarabicum (2n = 2x = 14, EbEb or JbJb or JJ), is a well-known alien source of salinity tolerance and disease resistance for wheat improvement. The true genetic potential and effect of such introgressions into wheat can be best studied in chromosomal addition or substitution lines. Here, we report the generation and characterization of various categories of Th. bessarabicum derivatives in both hexaploid and tetraploid cultivated wheats. Sequential non-denaturing fluorescence in situ hybridization (ND-FISH) and genomic in situ hybridization (GISH) are robust techniques to visualize the size of alien introgressions and breakpoints. We identified a complete set of monosomic addition lines into both bread wheat and durum wheat, except for 7J in durum wheat, by sequential ND-FISH and GISH. We also characterized alien derivatives belonging to various classes including mono-telosomic additions, disomic additions, monosomic substitutions, double monosomic substitutions, monosomic substitution-monosomic additions, double monosomic additions, and multiple monosomic additions into both bread and durum wheats. In addition, various wheat-Th. bessarabicum recombinant chromosomes were also detected in six alien derivatives. These wheat-Th. bessarabicum derivatives will provide useful cytogenetic resources for improvement of both hexaploid and tetraploid wheats.

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Triacylglycerol is a major component of vegetable oil in seeds and fruits of many plants, but its production in vegetative tissues is rather limited. It would be intriguing and important to explore any possibility to expand current oil production platforms, for example from the plant vegetative tissues. By expressing a suite of transgenes involved in the triacylglycerol biosynthesis, we have previously observed substantial accumulation of triacylglycerol in tobacco (Nicotiana tabacum) leaf and potato (Solanum tuberosum) tuber. In this study, simultaneous RNA interference (RNAi) downregulation of ADP-glucose pyrophosphorylase (AGPase) and Sugar-dependent1 (SDP1), was able to increase the accumulation of triacylglycerol and other lipids in both wild type potato and the previously generated high oil potato line 69. Particularly, a 16-fold enhancement of triacylglycerol production was observed in the mature transgenic tubers derived from the wild type potato, and a two-fold increase in triacylglycerol was observed in the high oil potato line 69, accounting for about 7% of tuber dry weight, which is the highest triacylglycerol accumulation ever reported in potato. In addition to the alterations of lipid content and fatty acid composition, sugar accumulation, starch content of the RNAi potato lines in both tuber and leaf tissues were also substantially changed, as well as the tuber starch properties. Microscopic analysis further revealed variation of lipid droplet distribution and starch granule morphology in the mature transgenic tubers compared to their parent lines. This study reflects that the carbon partitioning between lipid and starch in both leaves and non-photosynthetic tuber tissues, respectively, are highly orchestrated in potato, and it is promising to convert low-energy starch to storage lipids via genetic manipulation of the carbon metabolism pathways.

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We build on results from our previous investigation into ice Ih using a combination of classical many-body molecular dynamics (MB-MD) and normal mode (NM) calculations to obtain molecular-level information on the spectroscopic signatures in the OH stretching region for all seven of the known ordered crystalline ice phases. The classical MB-MD spectra are shown to capture the important spectral features by comparing with experimental Raman spectra. This motivates the use of the classical simulations in understanding the spectral features of the various ordered ice phases in molecular terms. This is achieved through NM analysis to first demonstrate that the MB-MD spectra can be well recovered through the transition dipole moments and polarizability tensors calculated from each NM. From the normal mode calculations, measures of the amount of symmetric and antisymmetric stretching are calculated for each ice, as well as an approximation of how localized each mode is. These metrics aid in viewing the ice phases on a continuous spectrum determined by their density. As in ice Ih, it is found that most of the other ordered ice phases have highly delocalized modes and their spectral features cannot, in general, be described in terms of molecular normal modes. The lone exception is ice VIII, the densest crystalline ice phase. Despite being found only at high pressure, the symmetry index shows a clear separation of symmetric and antisymmetric stretching modes, giving rise to two distinct features.

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Clutch size is a key avian fitness and life history trait. A physiological model for clutch size determination (CSD), involving an anti-gonadal effect of prolactin (PRL) via suppression of luteinizing hormone (LH), was proposed over 20 years ago, but has received scant experimental attention since. The few studies looking at a PRL-based mechanistic hypothesis for CSD have been equivocal, but recent experiments utilizing a pharmacological agent to manipulate PRL in the zebra finch (Taeniopygia guttata) found no support for a role of this hormone in clutch size determination. Here, we take a complementary approach by manipulating clutch size through egg removal, examining co-variation in PRL and LH between two breeding attempts, as well as through experimentally-extended laying. Clutch size increased for egg removal females, but not controls, but this was not correlated with changes in PRL or LH. There were also no differences in PRL between egg removal females and controls, nor did PRL levels during early, mid- or late-laying of supra-normal clutches predict clutch size. By uncoupling PRL, LH and clutch size in our study, several key predictions of the PRL-based mechanistic model for CSD were not supported. However, a positive correlation between PRL levels late in laying and days relative to the last egg (clutch completion) provides an alternative explanation for the equivocal results surrounding the conventional PRL-based physiological model for CSD. We suggest that females coordinate PRL-mediated incubation onset with clutch completion to minimize hatching asynchrony and sibling hierarchy, a behavior that is amplified in females laying larger clutches.

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BACKGROUND: A major step towards the success of chickens as a domesticated species was the separation between maternal care and reproduction. Artificial incubation replaced the natural maternal behaviour of incubation and, thus, in certain breeds, it became possible to breed chickens with persistent egg production and no incubation behaviour; a typical example is the White Leghorn strain. Conversely, some strains, such as the Silkie breed, are prized for their maternal behaviour and their willingness to incubate eggs. This is often colloquially known as broodiness. RESULTS: Using an F2 linkage mapping approach and a cross between White Leghorn and Silkie chicken breeds, we have mapped, for the first time, genetic loci that affect maternal behaviour on chromosomes 1, 5, 8, 13, 18 and 19 and linkage group E22C19W28. Paradoxically, heterozygous and White Leghorn homozygous genotypes were associated with an increased incidence of incubation behaviour, which exceeded that of the Silkie homozygotes for most loci. In such cases, it is likely that the loci involved are associated with increased egg production. Increased egg production increases the probability of incubation behaviour occurring because egg laying must precede incubation. For the loci on chromosomes 8 and 1, alleles from the Silkie breed promote incubation behaviour and influence maternal behaviour (these explain 12 and 26% of the phenotypic difference between the two founder breeds, respectively). CONCLUSIONS: The over-dominant locus on chromosome 5 coincides with the strongest selective sweep reported in chickens and together with the loci on chromosomes 1 and 8, they include genes of the thyrotrophic axis. This suggests that thyroid hormones may play a critical role in the loss of incubation behaviour and the improved egg laying behaviour of the White Leghorn breed. Our findings support the view that loss of maternal incubation behaviour in the White Leghorn breed is the result of selection for fertility and egg laying persistency and against maternal incubation behaviour.

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Clutch size is a fundamental predictor of avian fitness, widely-studied from evolutionary and ecological perspectives, but surprisingly little is known about the physiological mechanisms regulating clutch size variation. The only formal mechanistic hypothesis for avian clutch-size determination predicts an anti-gonadal effect of circulating prolactin (PRL) via the inhibition of luteinizing hormone (LH), and has become widely-accepted despite little experimental support. Here we investigated the relationship between pre-breeding and breeding plasma PRL and LH and clutch-size in captive-breeding female zebra finches (Taeniopygia guttata). Using a repeated-measures design, we followed individual females from pre-breeding, through multiple breeding attempts, and attempted to decrease PRL using the D2-receptor agonist, bromocriptine. Clutch size was independent of variation in pre-breeding PRL or LH, although pre-breeding LH was negatively correlated with the time between pairing and the onset of laying. Clutch size was independent of variation in plasma PRL on all days of egg-laying. Bromocriptine treatment had no effect on plasma PRL, but in this breeding attempt clutch size was also independent of plasma PRL. Finally, we found no evidence for an inverse relationship between plasma PRL and LH levels, as predicted if PRL had inhibitory effects via LH. Thus, our data fail to provide any support for the involvement of circulating PRL in clutch size determination. These findings suggest that alternative models for hormonal control of avian clutch size need to be considered, perhaps involving downstream regulation of plasma PRL at the level of the ovary, or other hormones that have not been considered to date.

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In mammals Orexin-A and -B are neuropeptides involved in the hypothalamic regulation of diverse physiological functions including food intake and the sleep-wake cycle. This generalisation was investigated in meat-(broiler) and layer-type juvenile domestic chickens by immunocytochemical localisation of orexin A/B in the hypothalamus, and by measurements of hypothalamic hypocretin mRNA which encodes for orexin A/B after chronic food restriction, and during the sleep-wake cycle. Orexin immunoreactive fibres were observed throughout the hypothalamus with cell bodies in and around the paraventricular nucleus. No differences were observed in the pattern of immunoreactivity using anti- human orexin-A, or -B antisera. The amount of hypothalamic hypocretin mRNA in food -restricted broilers was higher than in broilers fed ad libitum, but the same as in layer- type hens fed ad libitum. Hypothalamic hypocretin mRNA was increased (P<0.01) in 12-week-old broilers fed 25% of their ad libitum intake between 6-12 weeks of age. No difference in hypothalamic hypocretin mRNA was seen in 12-week-old layer- type hens when they were awake (1-2h after lights on) or sleeping (1-2h after lights off). It is concluded that in the chicken, we could not find evidence that hypothalamic orexin plays a role in the sleep-wake cycle and it may be involved in aspects of energy balance.

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Many bird species have advanced their seasonal timing in response to global warming, but we still know little about the causal effect of temperature. We carried out experiments in climate-controlled aviaries to investigate how temperature affects luteinizing hormone, prolactin, gonadal development, timing of egg laying and onset of moult in male and female great tits. We used both natural and artificial temperature patterns to identify the temperature characteristics that matter for birds. Our results show that temperature has a direct, causal effect on onset of egg-laying, and in particular, that it is the pattern of increase rather than the absolute temperature that birds use. Surprisingly, the pre-breeding increases in plasma LH, prolactin and in gonadal size are not affected by increasing temperature, nor do they correlate with the onset of laying. This suggests that the decision to start breeding and its regulatory mechanisms are fine-tuned by different factors. We also found similarities between siblings in the timing of both the onset of reproduction and associated changes in plasma LH, prolactin and gonadal development. In conclusion, while temperature affects the timing of egg laying, the neuroendocrine system does not seem to be regulated by moderate temperature changes. This lack of responsiveness may restrain the advance in the timing of breeding in response to climate change. But as there is heritable genetic variation on which natural selection can act, microevolution can take place, and may represent the only way to adapt to a warming world.

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In vertebrates, reproductive output often increases with age. Unlike older birds, first-year photoperiodic birds lack experience with the reproductively stimulatory effects of long day lengths (photostimulation). We examined whether age-related differences in annual reproductive development could be partially attributed to previous experience with photostimulation in the photoperiodic house finch (Carpodacus mexicanus). By manipulating photoperiod, we generated 2 groups of first-year females: a photo-experienced group that underwent 1 photoperiodically induced cycle of gonadal development and regression and a photo-naïve group exposed to long days since hatch. We transferred both groups from long to short days and then photostimulated and exposed them to male birdsong prior to sacrifice. Following concurrent photostimulation, both groups exhibited similar plasma luteinizing hormone surges and hypothalamic vasoactive intestinal polypeptide immunoreactivity. In contrast, hypothalamic gonadotropin-releasing hormone immunoreactivity and circulating vitellogenin levels were higher in photo-experienced birds, and yolk deposition occurred in only 2 females, both of which were photo-experienced. Our results demonstrate that photo-experience enhances some aspects of early photo-induced reproductive development and raise the hypothesis that photo-experience may account for at least some age-related variation in reproductive output.

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Most animals reproduce seasonally. They time their reproduction in response to environmental cues, like increasing photoperiod and temperature, which are predictive for the time of high food availability. Although individuals of a population use the same cues, they vary in their onset of reproduction, with some animals reproducing consistently early or late. In avian research, timing of reproduction often refers to the laying date of the first egg, which is a key determinant of fitness. Experiments measuring temporal patterns of reproductive hormone concentrations or gonadal size under controlled conditions in response to a cue commonly assume that these proxies are indicative of the timing of egg laying. This assumption often remains untested, with few studies reporting both reproductive development and the onset of laying. We kept in total 144 pairs of great tits (Parus major) in separate climate-controlled aviaries over 4 years to correlate pre-breeding plasma luteinizing hormone (LH), prolactin (PRL) and gonadal growth with the timing of laying. Individuals varied consistently in hormone concentrations over spring, but this was not directly related to the timing of gonadal growth, nor with the laying date of the first egg. The timing of gonadal development in both sexes was similarly not correlated with the timing of laying. This demonstrates the female's ability to adjust the onset of laying to environmental conditions irrespective of substantial differences in pre-laying development. We conclude that stages of reproductive development are regulated by different cues, and therefore egg laying dates need to be studied to measure the influences of environmental cues on timing of seasonal reproduction.

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The Challenge Hypothesis postulates that male vertebrates can respond to social challenges, such as simulated territorial intrusions, by rapidly increasing their concentrations of plasma androgens, such as testosterone (T). This increase may facilitate the expression of aggressive behavior and lead to persistence of this behavior even after withdrawal of the challenge, thus potentially promoting territoriality and the probability of winning future challenges. The scope of the Challenge Hypothesis was tested by exposing free-ranging male Cassin's Sparrows, Peucaea cassinii, to conspecific song playback (SPB) at the beginning of the vernal nesting season. Exposure to SPB stimulated aggressive behavior but did not influence plasma T. Furthermore, plasma T did not correlate with the duration of exposure to SPB, and the behavioral response to SPB did not differ in males that were challenged a second time shortly after the first challenge. As birds were investigated at a stage of their reproductive cycle when plasma T is presumably seasonally high due to photostimulation, the lack of hormonal response to SPB may have been due to the hypothalamus-pituitary-gonadal axis secreting hormones at maximum rates. This was not the case, however, because administration of gonadotropin-releasing hormone I rapidly stimulated the secretion of luteinizing hormone (LH) and T, and treatment with ovine LH rapidly stimulated T secretion.

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Chronic stress generally inhibits the activity of the reproductive system. Acute stress also is often inhibitory, but the mechanism involved and its persistence of action once animals are no longer exposed to the stressor are poorly understood. We investigated the effect of capture and restraint stress on plasma testosterone (T), luteinizing hormone (LH), and corticosterone (CORT) in free-ranging male rufous-winged sparrows, Peucaea carpalis. Stress decreased plasma T between 10 and 30 min after capture and restraint but did not influence plasma LH, the main hormone that controls T secretion, suggesting that stress did not decrease plasma T by inhibiting LH secretion. The stress-induced decrease in plasma T was associated with elevated plasma CORT, but there was no evidence that these effects were functionally related. Plasma stress-induced T was positively related to plasma initial T measured within 2 min of capture. This relationship was, however, complex as plasma T decreased proportionally more in response to stress in sparrows with high than low plasma initial T. The relative sensitivity to the same stressor was, therefore, individually variable and this variation was related to initial plasma T. Birds caught and restrained for 30 min, and then released on their breeding territory before recapture up to 6 h later, maintained depressed plasma T, indicating that the effect of acute stress on this hormone persists after the stressor removal. These studies provide new information on the effects of acute stress on plasma T in free-ranging birds. In particular, they are among the first to characterize the time course and to describe the persistence of these effects. The findings also contribute to identifying factors that are associated with individual differences in plasma hormone levels.

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Gonadotropin-releasing hormone (GnRH) is central to the control of vertebrate reproductive cycles and since GnRH orthologs are also present in invertebrates, it is likely that the common ancestor of bilateral animals possessed a GnRH-like peptide. In order to understand the evolutionary and comparative biology of GnRH peptides we cloned the cDNA transcripts of prepro GnRH-like peptides from two species of bivalve molluscs, the Yesso scallop Patinopecten yessoensis and the Pacific oyster Crassostrea gigas. We compared their deduced uncleaved and mature amino acid sequences with those from other invertebrates and vertebrates, and determined their sites of expression and biological activity. The two molluscan GnRH sequences increased the number of known protostome GnRHs to six different forms, indicating the current classification of protostome GnRHs requires further revision. In both molluscs, RT-PCR analysis showed that the genes were highly expressed in nervous tissue with lower levels present in peripheral tissues including the gonads, while immunocytochemistry, using anti-octopus GnRH-like peptide, demonstrated the presence of GnRH-like peptide in neural tissue. Putative scallop GnRH-like peptide stimulated spermatogonial cell division in cultured scallop testis, but the scallop GnRH-like peptide did not stimulate LH release from cultured quail pituitary cells. This is the first report of the cloning of bivalve GnRH-like peptide genes and of molluscan GnRH-like peptides that are biologically active in molluscs, but not in a vertebrate.

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Timing of reproduction in temperate-zone birds is strongly correlated with spring temperature, with an earlier onset of breeding in warmer years. Females adjust their timing of egg laying between years to be synchronized with local food sources and thereby optimize reproductive output. However, climate change currently disrupts the link between predictive environmental cues and spring phenology. To investigate direct effects of temperature on the decision to lay and its genetic basis, we used pairs of great tits (Parus major) with known ancestry and exposed them to simulated spring scenarios in climate-controlled aviaries. In each of three years, we exposed birds to different patterns of changing temperature. We varied the timing of a temperature change, the daily temperature amplitude, and the onset and speed of a seasonal temperature rise. We show that females fine-tune their laying in response to a seasonal increase in temperature, whereas mean temperature and daily temperature variation alone do not affect laying dates. Luteinizing hormone concentrations and gonadal growth in early spring were not influenced by temperature or temperature rise, possibly posing a constraint to an advancement of breeding. Similarities between sisters in their laying dates indicate genetic variation in cue sensitivity. These results refine our understanding of how changes in spring climate might affect the mismatch in avian timing and thereby population viability.

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In mammals, photoreception is restricted to cones, rods and a subset of retinal ganglion cells. By contrast, non-mammalian vertebrates possess many extraocular photoreceptors but in many cases the role of these photoreceptors and their underlying photopigments is unknown. In birds, deep brain photoreceptors have been shown to sense photic changes in daylength (photoperiod) and mediate seasonal reproduction. Nonetheless, the specific identity of the opsin photopigment 'sensor' involved has remained elusive. Previously, we showed that vertebrate ancient (VA) opsin is expressed in avian hypothalamic neurons and forms a photosensitive molecule. However, a direct functional link between VA opsin and the regulation of seasonal biology was absent. Here, we report the in vivo and in vitro absorption spectra (λ(max) = ~490 nm) for chicken VA photopigments. Furthermore, the spectral sensitivity of these photopigments match the peak absorbance of the avian photoperiodic response (λ(max) = 492 nm) and permits maximum photon capture within the restricted light environment of the hypothalamus. Such a correspondence argues strongly that VA opsin plays a key role in regulating seasonal reproduction in birds.

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Many seasonal breeders adjust the timing of reproduction in response to year-to-year variations in supplementary environmental cues, amongst which ambient temperature is thought to be most influential. However, it is possible that for species such as the great tit (Parus major L.), phenological cues from sprouting vegetation and the consequent abundance of invertebrate prey, although dependent on temperature, may provide supplementary environmental cues per se. This hypothesis was investigated in breeding pairs of great tits kept in outdoor aviaries. In spring, experimental pairs were provided with access to leafing birch branches and caterpillars as a visual food cue, while control pairs were provided with non-leafing branches. Observations were made on the onset of laying and on concentrations of plasma luteinizing hormone (LH) at regular intervals to monitor changes in reproductive function. The onset of egg laying was not advanced by the presence of leafing branches and caterpillars. LH concentrations increased during the course of the study, but phenological cues did not affect plasma LH levels in females and males. Early spring vegetation, such as the leafing of birch branches, and the appearance of caterpillar prey do not appear to play a significant role in fine-tuning the onset of egg laying in great tits.

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Segregating F(3) families, derived from a cross between oat cultivar Swan and the putative single gene line PC68, were used to determine the association of seed storage protein loci and resistance gene analogues (RGAs) with the crown rust resistance gene Pc68. SDS-PAGE analysis detected three avenin loci, AveX, AveY, and AveZ, closely linked to Pc68. Their diagnostic alleles are linked in coupling to Pc68 and were also detected in three additional lines carrying Pc68. Another protein locus was linked in repulsion to Pc68. In complementary studies, three wheat RGA clones (W2, W4, and W10) detected restriction fragment length polymorphisms (RFLPs) between homozygous resistant and homozygous susceptible F(3) DNA bulks. Four oat homologues of W2 were cloned and sequenced. RFLPs detected with two of them were mapped using F(3) and F(4) populations. Clone 18 detected a locus, Orga2, linked in repulsion to Pc68. Clone 22 detected several RFLPs including Orga1 (the closest locus to Pc68) and three RGA loci (Orga22-2, Orga22-3, and Orga22-4) loosely linked to Pc68. The diagnostic RFLPs linked in coupling to Pc68 were detected by clone 22 in three additional oat lines carrying Pc68 and have potential utility in investigating and improving crown rust resistance of oat.

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