Xiaoming Yang

The draft genome of sweet orange (Citrus sinensis)

Oranges are an important nutritional source for human health and have immense economic value. Here we present a comprehensive analysis of the draft genome of sweet orange (Citrus sinensis). The assembled sequence covers 87.3% of the estimated orange genome, which is relatively compact, as 20% is composed of repetitive elements. We predicted 29,445 protein-coding genes, half of which are in the heterozygous state. With additional sequencing of two more citrus species and comparative analyses of seven citrus genomes, we present evidence to suggest that sweet orange originated from a backcross hybrid between pummelo and mandarin. Focused analysis on genes involved in vitamin C metabolism showed that GalUR, encoding the rate-limiting enzyme of the galacturonate pathway, is significantly upregulated in orange fruit, and the recent expansion of this gene family may provide a genomic basis. This draft genome represents a valuable resource for understanding and improving many important citrus traits in the future.

Genomic analyses of primitive, wild and cultivated citrus provide insights into asexual reproduction

The emergence of apomixis—the transition from sexual to asexual reproduction—is a prominent feature of modern citrus. Here we de novo sequenced and comprehensively studied the genomes of four representative citrus species. Additionally, we sequenced 100 accessions of primitive, wild and cultivated citrus. Comparative population analysis suggested that genomic regions harboring energy- and reproduction-associated genes are probably under selection in cultivated citrus. We also narrowed the genetic locus responsible for citrus polyembryony, a form of apomixis, to an 80-kb region containing 11 candidate genes. One of these, CitRWP, is expressed at higher levels in ovules of polyembryonic cultivars. We found a miniature inverted-repeat transposable element insertion in the promoter region of CitRWP that cosegregated with polyembryony. This study provides new insights into citrus apomixis and constitutes a promising resource for the mining of agriculturally important genes.

De novo transcriptome assembly of pummelo and molecular marker development.

Pummelo (Citrus grandis) is an important fruit crop worldwide because of its nutritional value. To accelerate the pummelo breeding program, it is essential to obtain extensive genetic information and develop relative molecular markers. Here, we obtained a 12-Gb transcriptome dataset of pummelo through a mixture of RNA from seven tissues using Illumina pair-end sequencing, assembled into 57,212 unigenes with an average length of 1010 bp. The annotation and classification results showed that a total of 39,584 unigenes had similar hits to the known proteins of four public databases, and 31,501 were classified into 55 Gene Ontology (GO) functional sub-categories. The search for putative molecular markers among 57,212 unigenes identified 10,276 simple sequence repeats (SSRs) and 64,720 single nucleotide polymorphisms (SNPs). High-quality primers of 1174 SSR loci were designed, of which 88.16% were localized to nine chromosomes of sweet orange. Of 100 SSR primers that were randomly selected for testing, 87 successfully amplified clear banding patterns. Of these primers, 29 with a mean PIC (polymorphic information content) value of 0.52 were effectively applied for phylogenetic analysis. Of the 20 SNP primers, 14 primers, including 54 potential SNPs, yielded target amplifications, and 46 loci were verified via Sanger sequencing. This new dataset will be a valuable resource for molecular biology studies of pummelo and provides reliable information regarding SNP and SSR marker development, thus expediting the breeding program of pummelo.

Genetic diversity and phylogenetic relationships of citron (Citrus medica L.) and its relatives in southwest China

Understanding the genetic diversity and phylogenetic relationship of citron (Citrus medica L.) is of great importance for making conservation and utilization strategies. Southeast Asia and its vicinity are usually regarded as one of the centers of origin for Citrus. The principal aims of this work were to evaluate the genetic diversity of 56 accessions of citron and its relatives mainly from southwest China, to investigate the genetic structures of citron populations, and to construct a phylogenetic tree to understand the evolution of some hybrids among citrons and other citrus types. SSR analysis detected a total of 387 alleles ranging from 2 to 12 alleles per locus, and nearly all accessions identified could be unequivocally distinguished. The observed and expected heterozygosities averaged 0.36 and 0.49, respectively. From a Bayesian cluster analysis, citrons were characterized by two distinguished genetic structures, which corresponded to the geographical distribution in southwest China. Citron and fingered citron might derive from a common ancestor. Based on the chloroplast sequences, the phylogenetic trees were constructed with congruent topologies and similar levels of statistical support for relationships among citron and its relatives using both maximum parsimony and Bayesian inference methods. Citron formed a monophyletic clade, which was completely different from mandarin (C. reticulata) and pummelo (C. grandis). The genetic contributors of some hybrid species, such as C. limonia, C. aurantifolia, and C. limon, were also discussed, and citron appeared to be a primary contributor to the speciation of these secondary citrus species.

Control of postharvest grey mould decay of nectarine by tea polyphenol combined with tea saponin.

The control efficacy of tea polyphenol (TP) in combination with tea saponin (TS) against nectarine grey mould decay caused by Botrytis cinerea and the underlying mechanism were investigated. The in vitro experiments showed that both TP and TS inhibited the mycelial growth in a dose‐dependent manner, and their combinations exhibited synergistic antifungal interactions with the synergistic ratios (SR) exceeding 1·5. The in vivo experiments showed that disease incidence and lesion diameter of grey mould of inoculated fruit were significantly lowered after being treated with the combination of TP and TS; furthermore, the activities of phenylalanine ammonia‐lyase (PAL), peroxidase (POD), polyphenol oxidase (PPO), chitinase and β‐1,3‐glucanase of inoculated fruit as well as the contents of total phenolic and lignin were significantly induced, the respiration rate of inoculated fruit was significantly decreased and therefore the quality decrease was accordingly retarded. These results revealed that TP in combination with TS could control grey mould of inoculated nectarines and their mechanism of action might be attributed to their active components, the induction of defensive system and the regulation of respiration.

Microsatellite polymorphism is likely involved in phytoene synthase activity in Citrus

Carotenoid content is an important determinant of pigmentation in citrus fruit. Phytoene synthase (PSY) is an important enzyme in the carotenoid synthesis pathway. In the present study, two similar but non-identical sequences transcripts (CsPsy1a and CsPsy1b) were isolated from sweet orange, and accumulated at a high level throughout the period of fruit maturation in both the peel and pulp. Although their transcript abundance was similar in the peel, Psy1a was the dominant form in the pulp. A functional analysis based on heterologous expression in Escherichia coli indicated that CsPsy1a was the more efficient converter of geranylgeranyl diphosphate to phytoene. In the pummelo cv. Gaophuang, which produces white fleshed fruit, Psy1b but not Psy1a transcript was detected. Expression of CsPsy1 genes and another seven carotenoid biosynthetic genes were studied in the pulp of ‘Anliu’ sweet orange and ‘Gaophuang’ pummelo, the variation in expression of genes associated with carotenoid synthesis could not explain the observed differences in carotenoid accumulation between orange and pummelo. Further site-directed mutagenesis in PSY1 suggested that maybe it was the number of AAT repeat units present in the gene which underlay variable activity of PSY1 in Citrus. These results have revealed the presence of diverse alleles of the Psy1 gene, with simple sequence repeats (SSRs) polymorphisms causing the change of enzymatic activity, are involved in the accumulation of total carotenoid in citrus fruits.

Molecular phylogeography and population evolution analysis of Citrus ichangensis (Rutaceae)

Ichang papeda (Citrus ichangensis), a wild and endemic perennial plant in Rutaceae, is characterized by the existence of wild and natural populations in southwestern and middle-west China. We analyzed a total of 231 individuals across 16 natural populations using chloroplast SSR markers, nuclear SSR markers, and single-copy nuclear genes. Standard population genetic analyses as well as Bayesian and maximum likelihood models were used to clarify the genetic diversity, population differentiation, barriers to gene flow, bottleneck events, isolation by distance, history migration, demographic history among populations, and phylogeny evolution. The chloroplast and nuclear genome analyses revealed a low level of genetic diversity in C. ichangensis. Clear signals of recent bottlenecks and strong patterns of isolation by distance were detected among different subpopulations, indicating a low extent of historical gene flow for this species and that genetic drift would occur after population differentiation. Bayesian clustering analyses revealed a clear pattern of genetic structure, with one cluster spanning the potential refugia in Wuling Mountains and Ta-pa Mountains, and other two clusters covering a more limited distribution range. The demographic history also supported the scenario that two isolated clusters originated in parallel from the genetic diversity center. Taxonomically, Ichang papeda may be a member of subgenus Citrus. Owing to the complicated topography, the mountainous regions and the Yangtze River have provided long-term stable habitats for C. ichangensis and acted as main barriers for its expansion, which might facilitate the process of speciation. Statistical population models and genetic data indicated strong genetic structure in C. ichangensis, which might result from the restricted gene flow, genetic drift, and population bottlenecks.

Integrated transcriptomic and metabolomic analyses of a wax deficient citrus mutant exhibiting jasmonic acid-mediated defense against fungal pathogens

AbstractNaturally, resistant crop germplasms are important resources for managing the issues of agricultural product safety and environment deterioration. We found a spontaneous mutant of ‘Newhall’ navel orange (Citrus sinensis Osbeck) (MT) with broad-spectrum protections against fungal pathogens in the orchard, postharvest-storage, and artificial inoculation conditions. To understand the defense mechanism of MT fruit, we constructed a genome-scale metabolic network that integrated metabolome and transcriptome datasets. The coordinated transcriptomic and metabolic data were enriched in two sub-networks, showing the decrease in very long chain fatty acid (by 41.53%) and cuticular wax synthesis (by 81.34%), and increase in the synthesis of jasmonic acid (JA) (by 95.23%) and JA-induced metabolites such as 5-dimethylnobietin (by 28.37%) in MT. Furthermore, cytological and biochemical analyses confirmed that the response to fungal infection in MT was independent of wax deficiency and was correlated with the levels of jasmonates, and the expression of plant defensin gene PDF1.2. Results of exogenous application of MeJA and JA inhibitors such as propyl gallate proved that JA-mediated defense contributes to the strong tolerance against pathogens in MT. Our results indicated that jasmonate biosynthesis and signaling are stimulated by the fatty acid redirection of MT, and participate in the tolerance of pathogenic fungi.Orange: Hormone protects mutant orange from fungal pathogensElevated hormone signaling underpins fungal protection in a naturally occurring variety of orange found in China. Yunjiang Cheng from Huazhong Agricultural 
University in Wuhan, China, and coworkers analyzed all the genes expressed and metabolites produced by a mutant variety of ‘Newhall’ navel orange (Citrus sinensis Osbeck). The researchers found that, compared to wild-type oranges, the fungus-resistant mutant showed a decrease in both fatty acid synthesis and surface wax production, as well as an increase in synthesis of jasmonic acid, a plant hormone involved in anti-microbial defenses. The mutant seems to redirect production of fatty acids to make more jasmonic acid—and it’s this hormone that mediates the plant’s strong tolerance to fungal pathogens. This information could help agronomists breed other varieties of pest-resistant oranges that require less chemical fungicide under field and storage conditions.