Mapping and Identifying Candidate Genes Enabling Cadmium Accumulation in Brassica napus Revealed by Combined BSA-Seq and RNA-Seq Analysis.

Rapeseed has the ability to absorb cadmium in the roots and transfer it to aboveground organs, making it a potential species for remediating soil cadmium (Cd) pollution. However, the genetic and molecular mechanisms underlying this phenomenon in rapeseed are still unclear. In this study, a 'cadmium-enriched' parent, 'P1', with high cadmium transport and accumulation in the shoot (cadmium root: shoot transfer ratio of 153.75%), and a low-cadmium-accumulation parent, 'P2', (with a cadmium transfer ratio of 48.72%) were assessed for Cd concentration using inductively coupled plasma mass spectrometry (ICP-MS). An F2 genetic population was constructed by crossing 'P1' with 'P2' to map QTL intervals and underlying genes associated with cadmium enrichment. Fifty extremely cadmium-enriched F2 individuals and fifty extremely low-accumulation F2 individuals were selected based on cadmium content and cadmium transfer ratio and used for bulk segregant analysis (BSA) in combination with whole genome resequencing. This generated a total of 3,660,999 SNPs and 787,034 InDels between these two segregated phenotypic groups. Based on the delta SNP index (the difference in SNP frequency between the two bulked pools), nine candidate Quantitative trait loci (QTLs) from five chromosomes were identified, and four intervals were validated. RNA sequencing of 'P1' and 'P2' in response to cadmium was also performed and identified 3502 differentially expressed genes (DEGs) between 'P1' and 'P2' under Cd treatment. Finally, 32 candidate DEGs were identified within 9 significant mapping intervals, including genes encoding a glutathione S-transferase (GST), a molecular chaperone (DnaJ), and a phosphoglycerate kinase (PGK), among others. These genes are strong candidates for playing an active role in helping rapeseed cope with cadmium stress. Therefore, this study not only sheds new light on the molecular mechanisms of Cd accumulation in rapeseed but could also be useful for rapeseed breeding programs targeting this trait.

Molecular Cues for Phenological Events in the Flowering Cycle in Avocado.

Reproductively mature horticultural trees undergo an annual flowering cycle that repeats each year of their reproductive life. This annual flowering cycle is critical for horticultural tree productivity. However, the molecular events underlying the regulation of flowering in tropical tree crops such as avocado are not fully understood or documented. In this study, we investigated the potential molecular cues regulating the yearly flowering cycle in avocado for two consecutive crop cycles. Homologues of flowering-related genes were identified and assessed for their expression profiles in various tissues throughout the year. Avocado homologues of known floral genes FT, AP1, LFY, FUL, SPL9, CO and SEP2/AGL4 were upregulated at the typical time of floral induction for avocado trees growing in Queensland, Australia. We suggest these are potential candidate markers for floral initiation in these crops. In addition, DAM and DRM1, which are associated with endodormancy, were downregulated at the time of floral bud break. In this study, a positive correlation between CO activation and FT in avocado leaves to regulate flowering was not seen. Furthermore, the SOC1-SPL4 model described in annual plants appears to be conserved in avocado. Lastly, no correlation of juvenility-related miRNAs miR156, miR172 with any phenological event was observed.

Breaking the tight genetic linkage between the a1 and sh2 genes led to the development of anthocyanin-rich purple-pericarp super-sweetcorn.

The existence of purple-pericarp super-sweetcorn based on the supersweet mutation, shrunken2 (sh2), has not been previously reported, due to its extremely tight genetic linkage to a non-functional anthocyanin biosynthesis gene, anthocyaninless1 (a1). Generally, pericarp-pigmented starchy purple corn contains significantly higher anthocyanin. The development of purple-pericarp super-sweetcorn is dependent on breaking the a1-sh2 tight genetic linkage, which occurs at a very low frequency of < 1 in 1000 meiotic crossovers. Here, to develop purple-pericarp super-sweetcorn, an initial cross between a male purple-pericarp maize, 'Costa Rica' (A1Sh2.A1Sh2) and a female white shrunken2 super-sweetcorn, 'Tims-white' (a1sh2.a1sh2), was conducted. Subsequent self-pollination based on purple-pericarp-shrunken kernels identified a small frequency (0.08%) of initial heterozygous F3 segregants (A1a1.sh2sh2) producing a fully sh2 cob with a purple-pericarp phenotype, enabled by breaking the close genetic linkage between the a1 and sh2 genes. Resulting rounds of self-pollination generated a F6 homozygous purple-pericarp super-sweetcorn (A1A1.sh2sh2) line, 'Tim1'. Genome sequencing revealed a recombination break between the a1 and yz1 genes of the a1-yz1-x1-sh2 multigenic interval. The novel purple-pericarp super-sweetcorn produced a similar concentration of anthocyanin and sugar as in its purple-pericarp maize and white super-sweetcorn parents, respectively, potentially adding a broader range of health benefits than currently exists with standard yellow/white sweetcorn.

Oxygen delivery in pediatric cardiac surgery and its association with acute kidney injury using machine learning.

OBJECTIVE: Acute kidney injury (AKI) after pediatric cardiac surgery with cardiopulmonary bypass (CPB) is a frequently reported complication. In this study we aimed to determine the oxygen delivery indexed to body surface area (Do2i) threshold associated with postoperative AKI in pediatric patients during CPB, and whether it remains clinically important in the context of other known independent risk factors. METHODS: A single-institution, retrospective study, encompassing 396 pediatric patients, who underwent heart surgery between April 2019 and April 2021 was undertaken. Time spent below Do2i thresholds were compared to determine the critical value for all stages of AKI occurring within 48 hours of surgery. Do2i threshold was then included in a classification analysis with known risk factors including nephrotoxic drug usage, surgical complexity, intraoperative data, comorbidities and ventricular function data, and vasoactive inotrope requirement to determine Do2i predictive importance. RESULTS: Logistic regression models showed cumulative time spent below a Do2i value of 350 mL/min/m2 was associated with AKI. Random forest models, incorporating established risk factors, showed Do2i threshold still maintained predictive importance. Patients who developed post-CPB AKI were younger, had longer CPB and ischemic times, and required higher inotrope support postsurgery. CONCLUSIONS: The present data support previous findings that Do2i during CPB is an independent risk factor for AKI development in pediatric patients. Furthermore, the data support previous suggestions of a higher threshold value in children compared with that in adults and indicate that adjustments in Do2i management might reduce incidence of postoperative AKI in the pediatric cardiac surgery population.

A haplotype resolved chromosomal level avocado genome allows analysis of novel avocado genes.

Avocado (Persea americana) is a member of the magnoliids, an early branching lineage of angiosperms that has high value globally with the fruit being highly nutritious. Here, we report a chromosome-level genome assembly for the commercial avocado cultivar Hass, which represents 80% of the world's avocado consumption. The DNA contigs produced from Pacific Biosciences HiFi reads were further assembled using a previously published version of the genome supported by a genetic map. The total assembly was 913 Mb with a contig N50 of 84 Mb. Contigs assigned to the 12 chromosomes represented 874 Mb and covered 98.8% of benchmarked single-copy genes from embryophytes. Annotation of protein coding sequences identified 48 915 avocado genes of which 39 207 could be ascribed functions. The genome contained 62.6% repeat elements. Specific biosynthetic pathways of interest in the genome were investigated. The analysis suggested that the predominant pathway of heptose biosynthesis in avocado may be through sedoheptulose 1,7 bisphosphate rather than via alternative routes. Endoglucanase genes were high in number, consistent with avocado using cellulase for fruit ripening. The avocado genome appeared to have a limited number of translocations between homeologous chromosomes, despite having undergone multiple genome duplication events. Proteome clustering with related species permitted identification of genes unique to avocado and other members of the Lauraceae family, as well as genes unique to species diverged near or prior to the divergence of monocots and eudicots. This genome provides a tool to support future advances in the development of elite avocado varieties with higher yields and fruit quality.

Tissue culture tools for selenium hyperaccumulator Neptunia amplexicaulis for development in phytoextraction.

Neptunia amplexicaulis is an herbaceous legume endemic to the Richmond area in central Queensland, Australia and is one of the strongest known Selenium hyperaccumulators on earth, showing significant potential to be utilised in Se phytoextraction applications. Here a protocol was established for in vitro micropropagation of Se hyperaccumulator N. amplexicaulis using nodal segments from in vitro-germinated seedlings. Shoot multiplication was achieved on Murashige and Skoog (MS) basal media supplemented with various concentrations of 6-Benzylaminopurine (BA) (1.0, 2.0, 3.0 mg L-1) alone or in combination with low levels of Naphthaleneacetic acid (NAA) (0.1, 0.2, 0.3 mg L-1), with 2.0 mg L-1 BA + 0.2 mg L-1 NAA found to be most effective. Elongated shoots were rooted in vitro using NAA, with highest root induction rate of 30% observed at 0.2 mg L-1 NAA. About 95% of the in vitro rooted shoots survived acclimatization. Clonally propagated plantlets were dosed with selenate/selenite solution and assessed for Se tissue concentrations using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) and found to retain their ability to hyperaccumulate. The protocol developed for this study has potential to be optimised for generating clonal plants of N. amplexicaulis for use in research and phytoextraction industry applications.

Quantitative Trait Loci for Heat Stress Tolerance in Brassica rapa L. Are Distributed across the Genome and Occur in Diverse Genetic Groups, Flowering Phenologies and Morphotypes.

Heat stress events during flowering in Brassica crops reduce grain yield and are expected to increase in frequency due to global climate change. We evaluated heat stress tolerance and molecular genetic diversity in a global collection of Brassica rapa accessions, including leafy, rooty and oilseed morphotypes with spring, winter and semi-winter flowering phenology. Tolerance to transient daily heat stress during the early reproductive stage was assessed on 142 lines in a controlled environment. Well-watered plants of each genotype were exposed to the control (25/15 °C day/night temperatures) or heat stress (35/25 °C) treatments for 7 d from the first open flower on the main stem. Bud and leaf temperature depression, leaf conductance and chlorophyll content index were recorded during the temperature treatments. A large genetic variation for heat tolerance and sensitivity was found for above-ground biomass, whole plant seed yield and harvest index and seed yield of five pods on the main stem at maturity. Genetic diversity was assessed on 212 lines with 1602 polymorphic SNP markers with a known location in the B. rapa physical map. Phylogenetic analyses confirmed two major genetic populations: one from East and South Asia and one from Europe. Heat stress-tolerant lines were distributed across diverse geographic origins, morphotypes (leafy, rooty and oilseed) and flowering phenologies (spring, winter and semi-winter types). A genome-wide association analysis of heat stress-related yield traits revealed 57 SNPs distributed across all 10 B. rapa chromosomes, some of which were associated with potential candidate genes for heat stress tolerance.

A Comprehensive High-Quality DNA and RNA Extraction Protocol for a Range of Cultivars and Tissue Types of the Woody Crop Avocado.

High-quality DNA and RNA forms the basis of genomic and genetic investigations. The extraction of DNA and RNA from woody trees, like avocado (Persea americana Mill.), is challenging due to compounds which interact with nucleic acids and influence separation. Previously reported methods of DNA and RNA extraction from avocado have issues of low yield, quality and applicability across different cultivars and tissue types. In the current study, methods have been optimised for high-quality DNA extraction from 40 avocado cultivars and RNA extraction from multiple tissue types, including roots, stem, leaves, flowers and fruits. The method is based on the modification of the cetyltrimethylammonium bromide buffer, centred around the specific optimisation of chemicals, such as sodium dodecyl sulphate, polyvinylpyrrolidone, sodium sulphite, polyethylene glycol and ß-mercaptoethanol. The DNA extraction method yielded high-molecular weight DNA from the leaf tissue of 40 avocado cultivars belonging to Mexican, Guatemalan and West Indian avocado horticultural groups. The method was further optimised for RNA extraction from different avocado plant parts, enabling extraction using amounts as low as ~10 mg of starting material. The DNA and RNA extracted was successfully used for long- and short-read sequencing and gene expression analysis. The methods developed may also be applicable to other recalcitrant plant species.

Cryopreservation of Woody Crops: The Avocado Case.

Recent development and implementation of crop cryopreservation protocols has increased the capacity to maintain recalcitrant seeded germplasm collections via cryopreserved in vitro material. To preserve the greatest possible plant genetic resources globally for future food security and breeding programs, it is essential to integrate in situ and ex situ conservation methods into a cohesive conservation plan. In vitro storage using tissue culture and cryopreservation techniques offers promising complementary tools that can be used to promote this approach. These techniques can be employed for crops difficult or impossible to maintain in seed banks for long-term conservation. This includes woody perennial plants, recalcitrant seed crops or crops with no seeds at all and vegetatively or clonally propagated crops where seeds are not true-to-type. Many of the world's most important crops for food, nutrition and livelihoods, are vegetatively propagated or have recalcitrant seeds. This review will look at ex situ conservation, namely field repositories and in vitro storage for some of these economically important crops, focusing on conservation strategies for avocado. To date, cultivar-specific multiplication protocols have been established for maintaining multiple avocado cultivars in tissue culture. Cryopreservation of avocado somatic embryos and somatic embryogenesis have been successful. In addition, a shoot-tip cryopreservation protocol has been developed for cryo-storage and regeneration of true-to-type clonal avocado plants.

Scion control of miRNA abundance and tree maturity in grafted avocado.

BACKGROUND: Grafting is the common propagation method for avocado and primarily benefits orchard production by reducing the time to tree productivity. It also allows use of scions and rootstocks specifically selected for improved productivity and commercial acceptance. Rootstocks in avocado may be propagated from mature tree cuttings ('mature'), or from seed ('juvenile'). While the use of mature scion material hastens early bearing/maturity and economic return, the molecular factors involved in the role of the scion and/or rootstock in early bearing/reduced juvenility of the grafted tree are still unknown. RESULTS: Here, we utilized juvenility and flowering associated miRNAs; miR156 and miR172 and their putative target genes to screen pre-graft and post-graft material in different combinations from avocado. The abundance of mature miR156, miR172 and the miR156 target gene SPL4, showed a strong correlation to the maturity of the scion and rootstock material in avocado. Graft transmissibility of miR156 and miR172 has been explored in annual plants. Here, we show that the scion may be responsible for grafted tree maturity involving these factors, while the rootstock maturity does not significantly influence miRNA abundance in the scion. We also demonstrate that the presence of leaves on cutting rootstocks supports graft success and contributes towards intergraft signalling involving the carbohydrate-marker TPS1. CONCLUSION: Here, we suggest that the scion largely controls the molecular 'maturity' of grafted avocado trees, however, leaves on the rootstock not only promote graft success, but can influence miRNA and mRNA abundance in the scion. This constitutes the first study on scion and rootstock contribution towards grafted tree maturity using the miR156-SPL4-miR172 regulatory module as a marker for juvenility and reproductive competence.

The avocado genome informs deep angiosperm phylogeny, highlights introgressive hybridization, and reveals pathogen-influenced gene space adaptation.

The avocado, Persea americana, is a fruit crop of immense importance to Mexican agriculture with an increasing demand worldwide. Avocado lies in the anciently diverged magnoliid clade of angiosperms, which has a controversial phylogenetic position relative to eudicots and monocots. We sequenced the nuclear genomes of the Mexican avocado race, P. americana var. drymifolia, and the most commercially popular hybrid cultivar, Hass, and anchored the latter to chromosomes using a genetic map. Resequencing of Guatemalan and West Indian varieties revealed that ∼39% of the Hass genome represents Guatemalan source regions introgressed into a Mexican race background. Some introgressed blocks are extremely large, consistent with the recent origin of the cultivar. The avocado lineage experienced 2 lineage-specific polyploidy events during its evolutionary history. Although gene-tree/species-tree phylogenomic results are inconclusive, syntenic ortholog distances to other species place avocado as sister to the enormous monocot and eudicot lineages combined. Duplicate genes descending from polyploidy augmented the transcription factor diversity of avocado, while tandem duplicates enhanced the secondary metabolism of the species. Phenylpropanoid biosynthesis, known to be elicited by Colletotrichum (anthracnose) pathogen infection in avocado, is one enriched function among tandems. Furthermore, transcriptome data show that tandem duplicates are significantly up- and down-regulated in response to anthracnose infection, whereas polyploid duplicates are not, supporting the general view that collections of tandem duplicates contribute evolutionarily recent "tuning knobs" in the genome adaptive landscapes of given species.

Juvenility and Vegetative Phase Transition in Tropical/Subtropical Tree Crops.

In plants, juvenile to adult phase transition is regulated by the sequential activity of two microRNAs: miR156 and miR172. A decline in miR156 and increase in miR172 abundance is associated with phase transition. There is very limited information on phase transition in economically important horticultural tree crops, which have a significantly long vegetative phase affecting fruit bearing. Here, we profiled various molecular cues known to be involved in phase transition and flowering, including the microRNAs miR156 and miR172, in three horticultural tree crops: avocado (Persea americana), mango (Mangifera indica), and macadamia (Macadamia integrifolia). We observed that miR156 expression decreases as these trees age and can potentially be used as a juvenility marker. Consistent with findings in annual plants, we also observed conserved regulation of the miR156-SPL3/4/5 regulatory module in these genetically distant tree crops, suggesting that this pathway may play a highly conserved role in vegetative identity. Meanwhile, the abundance of miR172 and its target AP2-like genes as well as the accumulation level of SPL9 transcripts were not related with plant age in these crops except in avocado where miR172 expression increased steadily. Finally, we demonstrate that various floral genes, including AP1 and SOC1 were upregulated in the reproductive phase and can be used as potential markers for the reproductive phase transition. Overall, this study provides an insight into the molecular associations of juvenility and phase transition in horticultural trees where crop breeding and improvement are encumbered by long juvenile phases.

Molecular characterization and analysis of conserved potyviral motifs in bean common mosaic virus (BCMV) for RNAi-mediated protection.

Australian bean common mosaic virus (BCMV) isolates were sequenced, and the sequences were compared to global BCMV and bean common mosaic necrosis virus (BCMNV) sequences and analysed for conserved potyviral motifs to generate in planta RNA-interference (RNAi) resistance. Thirty-nine out of 40 previously reported potyvirus motifs were conserved among all 77 BCMV/BCMNV sequences. Two RNAi target regions were selected for dsRNA construct design, covering 920 bp of the nuclease inclusion b (NIb) protein and 461 bp of the coat protein (CP). In silico prediction of the effectiveness of these constructs for broad-spectrum defence against the 77 BCMV and BCMNV sequences was done via analysis of putative 21-nucleotide (nt) and 22-nt small-interfering RNAs (siRNAs) generated from the target regions. The effectiveness of both constructs for siRNA generation and BCMV RNAi-mediated resistance was validated in Nicotiana benthamiana transient assays.

Identification and characterization of miRNAs in ripening fruit of Lycium barbarum L. using high-throughput sequencing.

MicroRNAs (miRNAs) are master regulators of gene activity documented to play central roles in fruit ripening in model plant species, yet little is known of their roles in Lycium barbarum L. fruits. In this study, miRNA levels in L. barbarum fruit samples at four developmental stages, were assayed using Illumina HiSeqTM2000. This revealed the presence of 50 novel miRNAs and 38 known miRNAs in L. barbarum fruits. Of the novel miRNAs, 36 were specific to L. barbarum fruits compared with L. chinense. A number of stage-specific miRNAs were identified and GO terms were assigned to 194 unigenes targeted by miRNAs. The majority of GO terms of unigenes targeted by differentially expressed miRNAs are "intracellular organelle," "binding," "metabolic process," "pigmentation," and "biological regulation." Enriched KEGG analysis indicated that nucleotide excision repair and ubiquitin mediated proteolysis were over-represented during the initial stage of ripening, with ABC transporters and sulfur metabolism pathways active during the middle stages and ABC transporters and spliceosome enriched in the final stages of ripening. Several miRNAs and their targets serving as potential regulators in L. barbarum fruit ripening were identified using quantitative reverse transcription polymerase chain reaction. The miRNA-target interactions were predicted for L. barbarum ripening regulators including miR156/157 with LbCNR and LbWRKY8, and miR171 with LbGRAS. Additionally, regulatory interactions potentially controlling fruit quality and nutritional value via sugar and secondary metabolite accumulation were identified. These include miR156 targeting of fructokinase and 1-deoxy-D-xylulose-5-phosphate synthase and miR164 targeting of beta-fructofuranosidase. In sum, valuable information revealed by small RNA sequencing in this study will provide a solid foundation for uncovering the miRNA-mediated mechanism of fruit ripening and quality in this nutritional food.

Microspore culture reveals complex meiotic behaviour in a trigenomic Brassica hybrid.

BACKGROUND: Development of synthetic allohexaploid Brassica (2n = AABBCC) would be beneficial for agriculture, as allelic contributions from three genomes could increase hybrid vigour and broaden adaptation. Microspore culture of a near-allohexaploid hybrid derived from the cross (B. napus × B. carinata) × B. juncea was undertaken in order to assess the frequency and distribution of homologous and homoeologous crossovers in this trigenomic hybrid. SNP and SSR molecular markers were used to detect inheritance of A, B and C genome alleles in microspore-derived (MD) progeny. SNP allele copy number was also assessed. The MD progeny were also compared to progeny derived by self-pollination and open-pollination for fertility (estimated by self-pollinated seed set and pollen viability) and DNA ploidy (measured by flow cytometry). RESULTS: In the trigenomic hybrid, homologous chromosome pairs A(j)-A(n), B(j)-B(c) and C(n)-C(c) had similar meiotic crossover frequencies and segregation to that previously observed in established Brassica species, as demonstrated by marker haplotype analysis of the MD population. Homoeologous pairing between chromosomes A1-C1, A2-C2 and A7-C6 was detected at frequencies of 12-18 %, with other homoeologous chromosome regions associating from 8 % (A3-C3) to 0-1 % (A8-C8, A8-C9) of the time. Copy number analysis revealed eight instances of additional chromosomes and 20 instances of chromosomes present in one copy in somatically doubled MD progeny. Presence of chromosome A6 was positively correlated with self-pollinated seed set and pollen viability in the MD population. Many MD progeny were unable to produce self-pollinated seed (76 %) or viable pollen (53 %), although one MD plant produced 198 self-pollinated seeds. Average fertility was significantly lower in progeny obtained by microspore culture than progeny obtained by self-pollination or open-pollination, after excluding MD progeny which had not undergone chromosome doubling. CONCLUSIONS: Based on SNP data analysis of the microspore-derived progeny, crossover frequency per chromosome in the allohexaploid hybrid was found to be similar to that in established Brassica species, suggesting that the higher chromosome number did not significantly disrupt cellular regulation of meiosis. SNP allele copy number analysis revealed the occurrence not only of homoeologous duplication/deletion events but also other cryptic duplications and deletions that may have been the result of mitotic instability. Microspore culture simplified the assessment of chromosome behaviour in the allohexaploid hybrid but yielded progeny with lower fertility and a greater range of ploidy levels compared to progeny obtained by self- or open-pollination.

Chromosome-level genome map provides insights into diverse defense mechanisms in the medicinal fungus Ganoderma sinense.

Fungi have evolved powerful genomic and chemical defense systems to protect themselves against genetic destabilization and other organisms. However, the precise molecular basis involved in fungal defense remain largely unknown in Basidiomycetes. Here the complete genome sequence, as well as DNA methylation patterns and small RNA transcriptomes, was analyzed to provide a holistic overview of secondary metabolism and defense processes in the model medicinal fungus, Ganoderma sinense. We reported the 48.96 Mb genome sequence of G. sinense, consisting of 12 chromosomes and encoding 15,688 genes. More than thirty gene clusters involved in the biosynthesis of secondary metabolites, as well as a large array of genes responsible for their transport and regulation were highlighted. In addition, components of genome defense mechanisms, namely repeat-induced point mutation (RIP), DNA methylation and small RNA-mediated gene silencing, were revealed in G. sinense. Systematic bioinformatic investigation of the genome and methylome suggested that RIP and DNA methylation combinatorially maintain G. sinense genome stability by inactivating invasive genetic material and transposable elements. The elucidation of the G. sinense genome and epigenome provides an unparalleled opportunity to advance our understanding of secondary metabolism and fungal defense mechanisms.

Molecular marker applications in plants.

Individuals within a population of a sexually reproducing species will have some degree of heritable genomic variation caused by mutations, insertion/deletions (INDELS), inversions, duplications, and translocations. Such variation can be detected and screened using molecular, or genetic, markers. By definition, molecular markers are genetic loci that can be easily tracked and quantified in a population and may be associated with a particular gene or trait of interest. This chapter will review the current major applications of molecular markers in plants.

Genome-wide identification of phenolic acid biosynthetic genes in Salvia miltiorrhiza.

MAIN CONCLUSION: Twenty-nine genes related to phenolic acid biosynthesis were identified in the Salvia miltiorrhiza genome. Nineteen of these are described for the first time, with ten genes experimentally correlating to phenolic acid biosynthesis. Vast stores of secondary metabolites exist in plants, many of which possess biological activities related to human health. Phenolic acid derivatives are a class of valuable bioactive pharmaceuticals abundant in the widely used Chinese medicinal herb, Salvia miltiorrhiza. The biosynthetic pathway for phenolic acids differs in this species from that of other investigated plants. However, the molecular basis for this is unknown, with systematic analysis of the genes involved not yet performed. As the first step towards unraveling this complex biosynthetic pathway in S. miltiorrhiza, the current genome assembly was searched for putatively involved genes. Twenty-nine genes were revealed, 19 of which are described here for the first time. These include 15 genes predicted in the phenylpropanoid pathway; seven genes in the tyrosine-derived pathway; six genes encoding putative hydroxycinnamoyltransferases, and one CYP98A, namely CYP98A78. The promoter regions, gene structures and expression patterns of these genes were examined. Furthermore, conserved domains and phylogenetic relationships with homologous proteins in other species were revealed. Most of the key enzymes, including 4-coumarate: CoA ligase, 4-hydroxyphenylpyruvate reductase and hydroxycinnamoyltransferase, were found in multiple copies, each exhibiting different characteristics. Ten genes putatively involved in rosmarinic acid biosynthesis are also described. These findings provide a foundation for further analysis of this complex and diverse pathway, with potential to enhance the synthesis of water-soluble medicinal compounds in S. miltiorrhiza.

Enhanced seed oil content by overexpressing genes related to triacylglyceride synthesis.

Oilseed rape (Brassica napus) is one of the most important oilseed crops globally. To meet increasing demand for oil-based products, the ability to enhance desirable oil content in the seed is required. This study assessed the capability of five genes in the triacylglyceride (TAG) synthesis pathway to enhance oil content. The genes BnGPDH, BnGPAT, BnDGAT, ScGPDH and ScLPAAT were overexpressed separately in a tobacco (Nicotiana benthamiana) model system, and simultaneously by pyramiding in B. napus, under the control of a seed specific Napin promoter. ScLPAAT transgenic plants showed a significant increase of 6.84% to 8.55% in oil content in tobacco seeds, while a ~4% increase was noted for BnGPDH and BnGPAT transgenic seeds. Seed-specific overexpression of all four genes in B. napus resulted in as high a 12.57% to 14.46% increased in seed oil content when compared to WT, equaling close to the sum of the single-gene overexpression increases in tobacco. Taken together, our study demonstrates that BnGPDH, BnGPAT and ScLPAAT may effectively increase seed oil content, and that simultaneous overexpression of these in transgenic B. napus may further enhance the desirable oil content relative to single-gene overexpressors.