Development and molecular cytogenetic characterization of Thinopyrum bessarabicum introgression lines in hexaploid and tetraploid wheats.

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.

Development of RNA-seq-based molecular markers for characterizing Thinopyrum bessarabicum and Secale introgressions in wheat.

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.

Genetic loci inherited from hens lacking maternal behaviour both inhibit and paradoxically promote this behaviour.

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.

Uncoupling clutch size, prolactin, and luteinizing hormone using experimental egg removal.

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.

Thyrotrophin in the pars tuberalis triggers photoperiodic response.

Molecular mechanisms regulating animal seasonal breeding in response to changing photoperiod are not well understood. Rapid induction of gene expression of thyroid-hormone-activating enzyme (type 2 deiodinase, DIO2) in the mediobasal hypothalamus (MBH) of the Japanese quail (Coturnix japonica) is the earliest event yet recorded in the photoperiodic signal transduction pathway. Here we show cascades of gene expression in the quail MBH associated with the initiation of photoinduced secretion of luteinizing hormone. We identified two waves of gene expression. The first was initiated about 14 h after dawn of the first long day and included increased thyrotrophin (TSH) beta-subunit expression in the pars tuberalis; the second occurred approximately 4 h later and included increased expression of DIO2. Intracerebroventricular (ICV) administration of TSH to short-day quail stimulated gonadal growth and expression of DIO2 which was shown to be mediated through a TSH receptor-cyclic AMP (cAMP) signalling pathway. Increased TSH in the pars tuberalis therefore seems to trigger long-day photoinduced seasonal breeding.

Circulating breeding and pre-breeding prolactin and LH are not associated with clutch size in the zebra finch (Taeniopygia guttata).

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.

Is microevolution the only emergency exit in a warming world? Temperature influences egg laying but not its underlying mechanisms in great tits.

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.

Increasing temperature, not mean temperature, is a cue for avian timing of reproduction.

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.

Up to the challenge? Hormonal and behavioral responses of free-ranging male Cassin's sparrows, Peucaea cassinii, to conspecific song playback.

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.

Vertebrate ancient opsin photopigment spectra and the avian photoperiodic response.

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.

Rapid stress-induced inhibition of plasma testosterone in free-ranging male rufous-winged sparrows, Peucaea carpalis: characterization, time course, and recovery.

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.

Individual variation in avian reproductive physiology does not reliably predict variation in laying date.

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.

Mollusc gonadotropin-releasing hormone directly regulates gonadal functions: a primitive endocrine system controlling reproduction.

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.

Hormone levels predict individual differences in reproductive success in a passerine bird.

Hormones mediate major physiological and behavioural components of the reproductive phenotype of individuals. To understand basic evolutionary processes in the hormonal regulation of reproductive traits, we need to know whether, and during which reproductive phases, individual variation in hormone concentrations relates to fitness in natural populations. We related circulating concentrations of prolactin and corticosterone to parental behaviour and reproductive success during both the pre-breeding and the chick-rearing stages in both individuals of pairs of free-living house sparrows, Passer domesticus. Prolactin and baseline corticosterone concentrations in pre-breeding females, and prolactin concentrations in pre-breeding males, predicted total number of fledglings. When the strong effect of lay date on total fledgling number was corrected for, only pre-breeding baseline corticosterone, but not prolactin, was negatively correlated with the reproductive success of females. During the breeding season, nestling provisioning rates of both sexes were negatively correlated with stress-induced corticosterone levels. Lastly, individuals of both sexes with low baseline corticosterone before and high baseline corticosterone during breeding raised the most offspring, suggesting that either the plasticity of this trait contributes to reproductive success or that high parental effort leads to increased hormone concentrations. Thus hormone concentrations both before and during breeding, as well as their seasonal dynamics, predict reproductive success, suggesting that individual variation in absolute concentrations and in plasticity is functionally significant, and, if heritable, may be a target of selection.

Spring phenology does not affect timing of reproduction in the great tit (Parus major).

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.

Genetic association of crown rust resistance gene Pc68, storage protein loci, and resistance gene analogues in oats.

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.

Seasonal changes in concentrations of plasma LH and prolactin associated with the advance in the development of photorefractoriness and molt by high temperature in the starling.

In a study on starlings (Sturnus vulgaris) kept on a simulated annual cycle in photoperiod, temperature had no effect on the timing or rate of testicular maturation but high temperature resulted in an advance in the timing of testicular regression and molt (Dawson, 2005). This study asks whether the earlier gonadal regression in response to higher temperature represents a central neuroendocrine response to temperature, and secondly, whether prolactin plays a role in the earlier regression. Castrated starlings were kept on a simulated annual cycle of photoperiod at either 8 or 18 degrees C. Circulating LH and prolactin concentrations were measured and the progress of the post-nuptial molt was recorded as an external indicator of the development of photorefractoriness. Additionally plasma prolactin was measured in samples taken from intact male and female starlings in the 2005 study. In castrated birds, LH concentrations decreased three weeks earlier at 18 degrees C. These birds also showed the same three week advance in molt as males and females in the earlier study. This demonstrates that the advance in regression caused by higher temperatures probably results from a central neuroendocrine mechanism, i.e., an advance in photorefractoriness, rather than an effect at the level of the gonads. Temperature had a highly significant effect on the changes in prolactin - peak prolactin occurred three weeks earlier at 18 degrees C. However, there was no clear consistent significant difference in prolactin between the two temperatures in advance of the onset of photorefractoriness, so the advance in photorefractoriness may not be mediated by prolactin. The higher temperature resulted in a significantly earlier decrease in prolactin and this may be causally related to the advance in molt.

Acute stress rapidly decreases plasma testosterone in a free-ranging male songbird: potential site of action and mechanism.

We used a free-ranging, seasonally breeding adult male songbird, the rufous-winged sparrow, Aimophila carpalis, to investigate the effects of acute stress-induced by capture followed by restraint, on the hypothalamo-pituitary-testicular axis. Intra- and interindividual comparisons revealed that males decreased their plasma testosterone (T) by 37-52% in response to acute stress. The decrease occurred within 15 min of capture and persisted for at least another 15 min. Within 15 min, the decrease in plasma T was not associated with a reduction in plasma luteinizing hormone (LH). Thirty minutes after capture and restraint, the decrease in plasma T either was likewise not associated with decreased plasma LH (intraindividual comparison) or concurred with a reduction in plasma LH (interindividual comparison). These observations indicate that effects of stress may have been mediated at the pituitary gland and also directly at the testicular levels. To address this question, we measured the hormonal response to an injection of the glutamate receptor agonist N-methyl-d,l-aspartate (NMA) to stimulate to stimulate the release of gonadotropin-releasing hormone (GnRH) or of GnRH to stimulate the release of LH. Treatment with NMA did not change plasma LH, presumably because the birds were in breeding condition and already secreting GnRH at a maximum rate. Administration of GnRH increased plasma LH equally in birds that were or were not stressed before the treatment. An injection of purified ovine LH (oLH) increased plasma T equally in birds that were or were not acutely stressed before the hormone injection. Thus, the observed acute stress-induced decrease in plasma T was apparently not mediated by decreased responsiveness of the pituitary gland to GnRH or of the testes to LH. Decreased plasma T following stress may involve a direct impairment of the testicular endocrine function.

Emmer wheat (Triticum dicoccon Schrank) improves water use efficiency and yield of hexaploid bread wheat.

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.

A Synergistic Genetic Engineering Strategy Induced Triacylglycerol Accumulation in Potato (Solanum tuberosum) Leaf.

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.