Identification and Validation of Key Genes Related to Preferred Flavour Profiles in Australian Commercial Papaya (Carica papaya L.).

Commercial papaya varieties grown in Australia vary greatly in taste and aroma. Previous profiling has identified undesirable 'off tastes' in existing varieties, discouraging a portion of the population from consuming papayas. Our focus on enhancing preferred flavours led to an exploration of the genetic mechanisms and biosynthesis pathways that underlie these desired taste profiles. To identify genes associated with consumer-preferred flavours, we conducted whole RNA sequencing and de novo genome assembly on papaya varieties RB1 (known for its sweet flavour and floral aroma) and 1B (less favoured due to its bitter taste and musty aroma) at both ripe and unripe stages. In total, 180,368 transcripts were generated, and 118 transcripts related to flavours were differentially expressed between the two varieties at the ripe stage. Five genes (cpBGH3B, cpPFP, cpSUS, cpGES and cpLIS) were validated through qPCR and significantly differentially expressed. These genes are suggested to play key roles in sucrose metabolism and aromatic compound production pathways, holding promise for future selective breeding strategies. Further exploration will involve assessing their potential across broader germplasm and various growth environments.

Transcriptomic stability or lability explains sensitivity to climate stressors in coralline algae.

BACKGROUND: Crustose coralline algae (CCA) are calcifying red macroalgae that play important ecological roles including stabilisation of reef frameworks and provision of settlement cues for a range of marine invertebrates. Previous research into the responses of CCA to ocean warming (OW) and ocean acidification (OA) have found magnitude of effect to be species-specific. Response to OW and OA could be linked to divergent underlying molecular processes across species. RESULTS: Here we show Sporolithon durum, a species that exhibits low sensitivity to climate stressors, had little change in metabolic performance and did not significantly alter the expression of any genes when exposed to temperature and pH perturbations. In contrast, Porolithon onkodes, a major coral reef builder, reduced photosynthetic rates and had a labile transcriptomic response with over 400 significantly differentially expressed genes, with differential regulation of genes relating to physiological processes such as carbon acquisition and metabolism. The differential gene expression detected in P. onkodes implicates possible key metabolic pathways, including the pentose phosphate pathway, in the stress response of this species. CONCLUSIONS: We suggest S. durum is more resistant to OW and OA than P. onkodes, which demonstrated a high sensitivity to climate stressors and may have limited ability for acclimatisation. Understanding changes in gene expression in relation to physiological processes of CCA could help us understand and predict how different species will respond to, and persist in, future ocean conditions predicted for 2100.

Biochemical, Sensory, and Molecular Evaluation of Flavour and Consumer Acceptability in Australian Papaya (Carica papaya L.) Varieties.

Inconsistency in flavour is one of the major challenges to the Australian papaya industry. However, objectively measurable standards of the compound profiles that provide preferable taste and aroma, together with consumer acceptability, have not been set. In this study, three red-flesh papayas (i.e., 'RB1', 'RB4', and 'Skybury') and two yellow-flesh papayas (i.e., '1B' and 'H13') were presented to a trained sensory panel and a consumer panel to assess sensory profiles and liking. The papaya samples were also examined for sugar components, total soluble solids, and 14 selected volatile compounds. Additionally, the expression patterns of 10 genes related to sweetness and volatile metabolism were assessed. In general, red papaya varieties had higher sugar content and tasted sweeter than yellow varieties, while yellow varieties had higher concentrations of citrus floral aroma volatiles and higher aroma intensity. Higher concentrations of glucose, linalool oxide, and terpinolene were significantly associated with decreased consumer liking. Significant differences were observed in the expression profiles of all the genes assessed among the selected papaya varieties. Of these, cpGPT2 and cpBGLU31 were positively correlated to glucose production and were expressed significantly higher in '1B' than in 'RB1' or 'Skybury'. These findings will assist in the strategic selective breeding for papaya to better match consumer and, hence, market demand.

Lens orientalis Contributes Quantitative Trait Loci and Candidate Genes Associated With Ascochyta Blight Resistance in Lentil.

Australian lentil production is affected by several major biotic constraints including Ascochyta blight (AB), caused by Ascochyta lentis, a devastating fungal disease. Cultivation of AB resistant cultivars, alongside agronomic management including fungicide application, is the current most economically viable control strategy. However, the breakdown of AB resistance in cultivars, such as Northfield and Nipper, suggests the need for introgression of new and diverse resistance genes. Successful introgression entails an understanding of the genetic basis of resistance. In this context, a biparental mapping population derived from a cross between a recently identified AB resistant accession ILWL 180 (Lens orientalis) and a susceptible cultivar ILL 6002 was produced. A genetic linkage map was constructed from single-nucleotide polymorphism markers generated using a genotyping-by-sequencing transcript approach. Genetic dissection of the mapping population revealed a major quantitative trait loci (QTL) region nested with three QTLs on linkage group 5 and explained 9.5-11.5 percent (%) of phenotypic variance for AB resistance. Another QTL was identified on LG2 with phenotypic variance of 9.6%. The identified QTL regions harbored putative candidate genes potentially associated with defense responses to A. lentis infection. The QTL analysis and the candidate gene information are expected to contribute to the development of diagnostic markers and enable marker-assisted resistance selection in lentil breeding programmes.

Papaya (Carica papaya L.) Flavour Profiling.

A major challenge to the papaya industry is inconsistency in fruit quality and, in particular, flavour, which is a complex trait that comprises taste perception in the mouth (sweetness, acidity, or bitterness) and aroma produced by several volatile compounds. Current commercial varieties vary greatly in their taste, likely due to historical prioritised selection for fruit appearance as well as large environmental effects. Therefore, it is important to better understand the genetic and biochemical mechanisms and biosynthesis pathways underpinning preferable flavour in order to select and breed for better tasting new commercial papaya varieties. As an initial step, objectively measurable standards of the compound profiles that provide papaya's taste and aroma, together with 'mouth feel', are required. This review presents an overview of the approaches to characterise the flavour profiles of papaya through sugar component determination, volatile compound detection, sensory panel testing, as well as genomics-based studies to identify the papaya flavour.

Current population structure and pathogenicity patterns of Ascochyta rabiei in Australia.

Ascochyta blight disease, caused by the necrotrophic fungus Ascochyta rabiei, is a major biotic constraint to chickpea production in Australia and worldwide. Detailed knowledge of the structure of the pathogen population and its potential to adapt to our farming practices is key to informing optimal management of the disease. This includes understanding the molecular diversity among isolates and the frequency and distribution of the isolates that have adapted to overcome host resistance across agroecologically distinct regions. Thanks to continuous monitoring efforts over the past 6 years, a comprehensive collection of A. rabiei isolates was collated from the major Australian chickpea production regions. To determine the molecular structure of the entire population, representative isolates from each collection year and growing region have been genetically characterized using a DArTseq genotyping-by-sequencing approach. The genotyped isolates were further phenotyped to determine their pathogenicity levels against a differential set of chickpea cultivars and genotype-phenotype associations were inferred. Overall, the Australian A. rabiei population displayed a far lower genetic diversity (average Nei's gene diversity of 0.047) than detected in other populations worldwide. This may be explained by the presence of a single mating-type in Australia, MAT1-2, limiting its reproduction to a clonal mode. Despite the low detected molecular diversity, clonal selection appears to have given rise to a subset of adapted isolates that are highly pathogenic on commonly employed resistance sources, and that are occurring at an increasing frequency. Among these, a cluster of genetically similar isolates was identified, with a higher proportion of highly aggressive isolates than in the general population. The discovery of distinct genetic clusters associated with high and low isolate pathogenicity forms the foundation for the development of a molecular pathotyping tool for the Australian A. rabiei population. Application of such a tool, along with continuous monitoring of the genetic structure of the population will provide crucial information for the screening of breeding material and integrated disease management packages.

Biosensor Technologies for Early Detection and Quantification of Plant Pathogens.

Plant pathogens are a major reason of reduced crop productivity and may lead to a shortage of food for both human and animal consumption. Although chemical control remains the main method to reduce foliar fungal disease incidence, frequent use can lead to loss of susceptibility in the fungal population. Furthermore, over-spraying can cause environmental contamination and poses a heavy financial burden on growers. To prevent or control disease epidemics, it is important for growers to be able to detect causal pathogen accurately, sensitively, and rapidly, so that the best practice disease management strategies can be chosen and enacted. To reach this goal, many culture-dependent, biochemical, and molecular methods have been developed for plant pathogen detection. However, these methods lack accuracy, specificity, reliability, and rapidity, and they are generally not suitable for in-situ analysis. Accordingly, there is strong interest in developing biosensing systems for early and accurate pathogen detection. There is also great scope to translate innovative nanoparticle-based biosensor approaches developed initially for human disease diagnostics for early detection of plant disease-causing pathogens. In this review, we compare conventional methods used in plant disease diagnostics with new sensing technologies in particular with deeper focus on electrochemical and optical biosensors that may be applied for plant pathogen detection and management. In addition, we discuss challenges facing biosensors and new capability the technology provides to informing disease management strategies.

Genome Sequence of the Fungus Nannizziopsis barbatae, an Emerging Reptile Pathogen.

Nannizziopsis barbatae is an emerging fungal pathogen capable of causing contagious dermatomycosis in reptiles. Here, we report a 31.54-Mb draft genome sequence of an isolate originating from an infected eastern water dragon in Brisbane, Australia.

Linkage mapping and quantitative trait loci analysis of sweetness and other fruit quality traits in papaya.

BACKGROUND: The identification and characterisation of quantitative trait loci (QTL) is an important step towards identifying functional sequences underpinning important crop traits and for developing accurate markers for selective breeding strategies. In this study, a genotyping-by-sequencing (GBS) approach detected QTL conditioning desirable fruit quality traits in papaya. RESULTS: For this, a linkage map was constructed comprising 219 single nucleotide polymorphism (SNP) loci across 10 linkage groups and covering 509 centiMorgan (cM). In total, 21 QTLs were identified for seven key fruit quality traits, including flesh sweetness, fruit weight, fruit length, fruit width skin freckle, flesh thickness and fruit firmness. Several QTL for flesh sweetness, fruit weight, length, width and firmness were stable across harvest years and individually explained up to 19.8% of the phenotypic variance of a particular trait. Where possible, candidate genes were proposed and explored further for their application to marker-assisted breeding. CONCLUSIONS: This study has extended knowledge on the inheritance and genetic control for key papaya physiological and fruit quality traits. Candidate genes together with associated SNP markers represent a valuable resource for the future of strategic selective breeding of elite Australian papaya cultivars.

Determination of the Key Resistance Gene Analogs Involved in Ascochyta rabiei Recognition in Chickpea.

Chickpea (Cicer arietinum L.) is an important cool season food legume, however, its production is severely constrained by the foliar disease Ascochyta blight caused by the fungus Ascochyta rabiei (syn. Phoma rabiei). Several disease management options have been developed to control the pathogen, including breeding for host plant resistance. However, the pathogen population is evolving to produce more aggressive isolates. For host resistance to be effective, the plant must quickly recognize the pathogen and instigate initial defense mechanisms, optimally at the point of contact. Given that the most resistant host genotypes display rapid pathogen recognition and response, the approach taken was to assess the type, speed and pattern of recognition via Resistance Gene Analog (RGA) transcription among resistant and susceptible cultivated chickpea varieties. RGAs are key factors in the recognition of plant pathogens and the signaling of inducible defenses. In this study, a suite of RGA loci were chosen for further investigation from both published literature and from newly mined homologous sequences within the National Center for Biotechnology Information (NCBI) database. Following their validation in the chickpea genome, 10 target RGAs were selected for differential expression analysis in response to A. rabiei infection. This was performed in a set of four chickpea varieties including two resistant cultivars (ICC3996 and PBA Seamer), one moderately resistant cultivar (PBA HatTrick) and one susceptible cultivar (Kyabra). Gene expression at each RGA locus was assessed via qPCR at 2, 6, and 24 h after A. rabiei inoculation with a previously characterized highly aggressive isolate. As a result, all loci were differentially transcribed in response to pathogen infection in at least one genotype and at least one time point after inoculation. Among these, the differential expression of four RGAs was significant and consistently increased in the most resistant genotype ICC3996 immediately following inoculation, when spore germination began and ahead of penetration into the plant's epidermal tissues. Further in silico analyses indicated that the differentially transcribed RGAs function through ADP-binding within the pathogen recognition pathway. These represent clear targets for future functional validation and potential for selective resistance breeding for introgression into elite cultivars.

Gonadal response of juvenile protogynous grouper (Epinephelus fuscoguttatus) to long-term recombinant follicle-stimulating hormone administration†.

The role of follicle-stimulating hormone (FSH) in the gonadal development of protogynous hermaphroditic grouper (Epinephelus fuscoguttatus) was investigated. Recombinant giant grouper (E. lanceolatus) FSH (rggFSH) was produced in yeast. Its receptor-binding capacity and steroidogenic potency were confirmed in vitro. Weekly injections of rggFSH to juvenile tiger grouper for 8 weeks (100 µg/kg body weight, BW) resulted in significantly larger and more advanced oocytes (cortical alveolar stage vs primary growth stage in control). Sustained treatment with rggFSH (20 to 38 weeks at 200 µg/kg BW) resulted in significant reduction in gonad size, degeneration of oocytes, and proliferation of spermatogonial cells, indicative of female to male sex change. Gene expression analysis showed that, while initiating female to male sex change, the rggFSH significantly suppressed the steroidogenic genes cyp11b, cyp19a1a, and foxl2 which restrained the endogenous production of sex steroid hormones and thus prevented the differentiation of spermatogonial cells. Expression profile of sex markers dmrt1, amh, figla, and bmp15 suggests that the observed sex change was restricted at the initiation stage. Based on these results, we propose that the process of female to male sex change in the protogynous grouper is initiated by FSH, rather than sex steroids, and likely involves steroid-independent pathway. The cortical alveolar stage in oocyte development is the critical point after which FSH-induced sex change is possible in grouper.

A transcriptomic investigation of digestive processes in orange-spotted grouper, Epinephelus coioides, before, during, and after metamorphic development.

The orange-spotted grouper (OG), Epinephelus coioides, is an ecologically and economically important species with strong market demand. However, larval rearing for this species is especially difficult, with mass mortality occurring at multiple stages including the period coinciding with metamorphic development. The aim of the present study was to characterise the molecular ontogenesis of genes that influence appetite, feeding, and digestion in OG larvae head and body tissue at 12, 18, and 50 days post hatch (dph), which coincides with the beginning and end of metamorphic development. The sequences of many transcripts involved in the regulation of appetite, feeding and digestive processes were detected from 12 dph in OG larvae, including those that were differentially expressed in body tissue in fish at different stages of development such as cholecystokinin, peptide Y, and meprin A. Of the transcripts encoding digestive enzymes, only the expression level of bile salt-activated lipase decreased as development progressed. In contrast, a dramatic increase in expression for other body-expressed transcripts encoding digestive enzymes and a proton pump subunit was observed at 50 dph, which is indicative of an increase in digestive capacity. In addition, we have provided evidence suggesting that various trypsinogen isoforms are present, and have differing expression patterns throughout larval development in whole body tissue. We also report on the presence of a prey-specific transcript encoding α-amylase that was present in the body-transcriptome. Taken together, these results give insight into the processes underpinning attainment of digestive capacity, and form the basis of a new transcriptomic database that will aid further study into the digestive development and dietary requirements of orange-spotted grouper larvae.

Transcriptome profiling of lentil (Lens culinaris) through the first 24 hours of Ascochyta lentis infection reveals key defence response genes.

BACKGROUND: Ascochyta blight, caused by the fungus Ascochyta lentis, is one of the most destructive lentil diseases worldwide, resulting in over $16 million AUD annual loss in Australia alone. The use of resistant cultivars is currently considered the most effective and environmentally sustainable strategy to control this disease. However, little is known about the genes and molecular mechanisms underlying lentil resistance against A. lentis. RESULTS: To uncover the genetic basis of lentil resistance to A. lentis, differentially expressed genes were profiled in lentil plants during the early stages of A. lentis infection. The resistant 'ILL7537' and susceptible 'ILL6002' lentil genotypes were examined at 2, 6, and 24 h post inoculation utilising high throughput RNA-Sequencing. Genotype and time-dependent differential expression analysis identified genes which play key roles in several functions of the defence response: fungal elicitors recognition and early signalling; structural response; biochemical response; transcription regulators; hypersensitive reaction and cell death; and systemic acquired resistance. Overall, the resistant genotype displayed an earlier and faster detection and signalling response to the A. lentis infection and demonstrated higher expression levels of structural defence-related genes. CONCLUSIONS: This study presents a first-time defence-related transcriptome of lentil to A. lentis, including a comprehensive characterisation of the molecular mechanism through which defence against A. lentis is induced in the resistant lentil genotype.

Genomic DNA variation confirmed Seriola lalandi comprises three different populations in the Pacific, but with recent divergence.

Captive breeding programs and aquaculture production have commenced worldwide for the globally distributed yellowtail kingfish (Seriola lalandi), and captive bred fingerlings are being shipped from the Southern Hemisphere to be farmed in the Northern Hemisphere. It was recently proposed that Pacific S. lalandi comprise at least three distinct species that diverged more than 2 million years ago. Here, we tested the hypothesis of different "species" in the Pacific using novel genomic data (namely single nucleotide polymorphisms and diversity array technology markers), as well as mtDNA and DNA microsatellite variation. These new data support the hypothesis of population subdivision between the Northeast Pacific, Northwest Pacific and South Pacific, and genetic divergence indicates restriction to the gene flow between hemispheres. However, our estimates of maximum mtDNA and nuclear DNA divergences of 2.43% and 0.67%, respectively, were within the ranges more commonly observed for populations within species than species within genera. Accordingly our data support the more traditional view that S. lalandi in the Pacific comprises three distinct populations rather than the subdivisions into several species.

Transcriptome analysis reveals differentially expressed genes associated with germ cell and gonad development in the Southern bluefin tuna (Thunnus maccoyii).

BACKGROUND: Controlling and managing the breeding of bluefin tuna (Thunnus spp.) in captivity is an imperative step towards obtaining a sustainable supply of these fish in aquaculture production systems. Germ cell transplantation (GCT) is an innovative technology for the production of inter-species surrogates, by transplanting undifferentiated germ cells derived from a donor species into larvae of a host species. The transplanted surrogates will then grow and mature to produce donor-derived seed, thus providing a simpler alternative to maintaining large-bodied broodstock such as the bluefin tuna. Implementation of GCT for new species requires the development of molecular tools to follow the fate of the transplanted germ cells. These tools are based on key reproductive and germ cell-specific genes. RNA-Sequencing (RNA-Seq) provides a rapid, cost-effective method for high throughput gene identification in non-model species. This study utilized RNA-Seq to identify key genes expressed in the gonads of Southern bluefin tuna (Thunnus maccoyii, SBT) and their specific expression patterns in male and female gonad cells. RESULTS: Key genes involved in the reproductive molecular pathway and specifically, germ cell development in gonads, were identified using analysis of RNA-Seq transcriptomes of male and female SBT gonad cells. Expression profiles of transcripts from ovary and testis cells were compared, as well as testis germ cell-enriched fraction prepared with Percoll gradient, as used in GCT studies. Ovary cells demonstrated over-expression of genes related to stem cell maintenance, while in testis cells, transcripts encoding for reproduction-associated receptors, sex steroids and hormone synthesis and signaling genes were over-expressed. Within the testis cells, the Percoll-enriched fraction showed over-expression of genes that are related to post-meiosis germ cell populations. CONCLUSIONS: Gonad development and germ cell related genes were identified from SBT gonads and their expression patterns in ovary and testis cells were determined. These expression patterns correlate with the reproductive developmental stage of the sampled fish. The majority of the genes described in this study were sequenced for the first time in T. maccoyii. The wealth of SBT gonadal and germ cell-related gene sequences made publicly available by this study provides an extensive resource for further GCT and reproductive molecular biology studies of this commercially valuable fish.

Assessment of yellowtail kingfish (Seriola lalandi) as a surrogate host for the production of southern bluefin tuna (Thunnus maccoyii) seed via spermatogonial germ cell transplantation.

Germ cell transplantation is an innovative technology for the production of interspecies surrogates, capable of facilitating easier and more economical management of large-bodied broodstock, such as the bluefin tuna. The present study explored the suitability of yellowtail kingfish (Seriola lalandi) as a surrogate host for transplanted southern bluefin tuna (Thunnus maccoyii) spermatogonial cells to produce tuna donor-derived gametes upon sexual maturity. Germ cell populations in testes of donor T. maccoyii males were described using basic histology and the molecular markers vasa and dead-end genes. The peripheral area of the testis was found to contain the highest proportions of dead-end-expressing transplantable Type A spermatogonia. T. maccoyii Type A spermatogonia-enriched preparations were transplanted into the coelomic cavity of 6-10-day-old post-hatch S. lalandi larvae. Fluorescence microscopy and polymerase chain reaction analysis detected the presence of tuna cells in the gonads of the transplanted kingfish fingerlings at 18, 28, 39 and 75 days after transplantation, indicating that the transplanted cells migrated to the genital ridge and had colonised the developing gonad. T. maccoyii germ cell-derived DNA or RNA was not detected at later stages, suggesting that the donor cells were not maintained in the hosts' gonads.

Small-scale capture, transport and tank adaptation of live, medium-sized Scombrids using "Tuna Tubes".

The transport of live fish is a crucial step to establish fish culture in captivity, and is especially challenging for species that have not been commonly cultured before, therefore transport and handling methods need to be optimized and tailored. This study describes the use of tuna tubes for small-scale transport of medium-sized pelagic fish from the Scombridae family. Tuna tubes are an array of vertical tubes that hold the fish, while fresh seawater is pumped up the tubes and through the fish mouth and gills, providing oxygen and removing wastes. In this study, 19 fish were captured using rod and line and 42 % of the captured fish were transported alive in the custom-designed tuna tubes to an on-shore holding tank: five mackerel tuna (Euthynnus affinis) and three leaping bonito (Cybiosarda elegans). Out of these, just three (15.8 % of total fish) acclimatized to the tank's condition. Based on these results, we discuss an improved design of the tuna tubes that has the potential to increase survival rates and enable a simple and low cost method of transporting of live pelagic fish.

Melanocortin receptor 1 and black pigmentation in the Japanese ornamental carp (Cyprinus carpio var. Koi).

Colors and their patterns are fascinating phenotypes with great importance for fitness under natural conditions. For this reason and because pigmentation is associated with diseases, much research was devoted to study the genetics of pigmentation in animals. Considerable contribution to our understanding of color phenotypes was made by studies in domesticated animals that exhibit dazzling variation in color traits. Koi strains, the ornamental variants of the common carp, are a striking example for color variability that was selected by man during a very short period on an evolutionary timescale. Among several pigmentation genes, genetic variation in Melanocrtin receptor 1 was repeatedly associated with dark pigmentation phenotypes in numerous animals. In this study, we cloned Melanocrtin receptor 1 from the common carp. We found that alleles of the gene were not associated with the development of black color in Koi. However, the mRNA expression levels of the gene were higher during dark pigmentation development in larvae and in dark pigmented tissues of adult fish, suggesting that variation in the regulation of the gene is associated with black color in Koi. These regulatory differences are reflected in both the timing of the dark-pigmentation development and the different mode of inheritance of the two black patterns associated with them. Identifying the genetic basis of color and color patterns in Koi will promote the production of this valuable ornamental fish. Furthermore, given the rich variety of colors and patterns, Koi serves as a good model to unravel pigmentation genes and their phenotypic effects and by that to improve our understanding of the genetic basis of colors also in natural populations.