High-density linkage maps for <Emphasis Type="Italic">Citrus sunki</Emphasis> and <Emphasis Type="Italic">Poncirus trifoliata</Emphasis> using DArTseq markers

The construction of a high-resolution genetic map of citrus would be of great value to breeders and to associate genomic regions with characteristics of agronomic interest. Here, we describe a novel high-resolution map of citrus using a population derived from a controlled cross between Citrus sunki (female parent) and Poncirus trifoliata (male parent). The genetic linkage maps were constructed using DArTseq markers and a pseudo-testcross strategy; only markers showing the expected segregation ratio were considered. To investigate synteny, all markers from both linkage maps were aligned with the genome of Citrus sinensis. The C. sunki map has a total of 2778 molecular markers and a size of 2446.6 cM, distributed across ten linkage groups. The map of P. trifoliata was built with 3084 markers distributed in a total of nine linkage groups, with a total size of 2411.6 cM. These maps are the most saturated linkage maps available for C. sunki and P. trifoliata and have high genomic coverage. We also demonstrated that the maps reported here are closely related to the reference genome of C. sinensis.     

Construction and characterization of a bacterial artificial chromosome library for <Emphasis Type="Italic">Camellia sinensis</Emphasis>

Tea is a popular and natural non-alcoholic beverage, and is produced from fresh leaves of Camellia sinensis. Tea leaves contain many bioactive compounds that have significant health benefits. We constructed a high quality bacterial artificial chromosome (BAC) library by using the fresh petals of C. sinensis “Shuchazao” for genome sequencing and improvement of genomic assembly. BAC library is still a significant tool for studies of functional genomes and preservation of precious genetic resources. The BAC library contains 161,280 clones with an average insert size of 113 kb, which represents approximately 6.2-fold coverage of haploid genome equivalents of C. sinensis. We characterized 20 complete BAC clones and 738 BAC end sequences (BESs) ranging from 105 to 917 bp. In addition, we predicted cis-regulatory elements of LAR (leucoanthocyanidin reductase), TCS (caffeine synthase), and TS (theanine synthetase) involved in tea characteristic metabolite synthesis and identified a larger number of light-responsive cis-acting elements in these three genes. Meanwhile, we analyzed alternative splicing of these three genes. Furthermore, 12 pairs of SSR primers were successfully amplified in tea plant DNA. The tea BAC library was a critical resource to accomplish de novo whole-genome sequencing, accelerate gene discovery and enhance molecular breeding of C. sinensis.     

The fitness of the cotton mealybug <Emphasis Type="Italic">Phenacoccus solenopsis</Emphasis> (Tinsley) is reduced after feeding on virus-infected <Emphasis Type="Italic">Hibiscus rosa</Emphasis>-<Emphasis Type="Italic">sinensis</Emphasis>

The cotton mealybug Phenacoccus solenopsis (Tinsley) and Cotton leaf curl Multan virus (CLCuMV), serious threats to economic crops and garden plants, have invaded southern China and widely infected Hibiscus rosa-sinensis. Whether an inter-species connection has facilitated the invasion process is unclear. In this study the interaction between P. solenopsis and H. rosa-sinensis infected with CLCuMV was investigated in the laboratory. We observed that 1st and 2nd instar nymphs of P. solenopsis preferred to feed on healthy H. rosa-sinensis leaves, whereas 3rd instar nymphs and female adults had no preference between healthy and virus-infected H. rosa-sinensis leaves. The developmental time of each P. solenopsis developmental stage increased significantly after feeding on infected H. rosa-sinensis leaves (p < 0.05). In particular, the development time for 2nd instar female and male nymphs and 3rd instar female nymphs increased by approximately twofold. The generation time of female mealybugs increased from 25.84 d on healthy H. rosa-sinensis to 32.12 d when feeding on CLCuMV-infected H. rosa-sinensis, and the survival rate decreased from 71.04 % on healthy H. rosa-sinensis to 5.80 % on infected plants. Nymph survival was most affected by feeding on infected plants. Additionally, the fecundity of female mealybugs feeding on infected H. rosa-sinensis decreased by 47.8 %. Thus, feeding on CLCuMV-infected H. rosa-sinensis significantly decreased the biological fitness and invading and colonizing abilities of P. solenopsis.     

Adaptive evolution of osmoregulatory-related genes provides insight into salinity adaptation in Chinese mitten crab, <Emphasis Type="Italic">Eriocheir sinensis</Emphasis>

Osmoregulation is an important mechanism by which euryhaline crustaceans regulate osmotic and ionic concentrations. The Chinese mitten crab (Eriocheir sinensis) is a strong osmoregulating animal model among crustacean species, as it can maintain its hemolymph composition and survives well in either seawater or freshwater. Osmoregulation by E. sinensis during physiological adaptation has been studied extensively. However, the genetic basis of osmoregulation in E. sinensis for acclimating to changing salinities remains unclear. The current study investigated five genes involved in E. sinensis osmoregulation and compared them with a representative marine crab Portunus trituberculatus to test whether adaptive evolution has occurred changing salinity conditions. The results showed that carbonic anhydrase (CA), cytochrome P450 4C (CYP4C), glutamate dehydrogenase (GDH), and the Na⁺/H⁺ exchanger (NHE) have undergone positive selection (i.e., directional selection) in E. sinensis. Thus, the positive selection in CA and NHE suggests that E. sinensis has enhanced capacity for maintaining systemic acid-base balance and ion regulation. GDH and CYP4C also demonstrated positive selection in E. sinensis, suggesting that E. sinensis might have acquired an enhanced capacity to metabolize glutamate and synthesize ecdysteroids in response to a change in osmotic concentration. The present study provides new insight into the molecular genetic basis of salinity adaption in E. sinensis.     

Prolonged culture of <Emphasis Type="Italic">Boesenbergia rotunda</Emphasis> cells reveals decreased growth and shoot regeneration capacity

We evaluated the ploidy levels and tissue culture responses of 16 Japanese Miscanthus accessions, which are registered and vegetatively maintained in the National Agriculture and Food Research Organization GeneBank, Japan, to screen suitable genotypes for the molecular breeding of Miscanthus species. A ploidy analysis showed that most M. sinensis and M. sinensis var. condensatus (var. condensatus) were putative diploids, but one accession identified as M. sinensis was unexpectedly a putative tetraploid. Additionally, M. sacchariflorus and its hybrid accessions were putative tetraploids. The deoxyribonucleic acid levels in var. condensatus were significantly higher than those in the diploid M. sinensis. Of the accessions, 10, including M. sinensis and var. condensatus, could induce plant regenerable embryogenic calli from apical meristems. We selected three of these M. sinensis accessions for further experiments because their calli growth rates were faster than those of the var. condensatus accessions. Tissue culture experiments with the selected accessions indicated that the frequencies of callus and green shoot formation strongly correlated with genotype. The broad-sense heritabilities of the embryogenic callus and green shoot formation frequencies in the selected accessions were 0.75 and 0.65, respectively, indicating that the cultures’ responses were mainly controlled by genetic factors. Thus, we further selected one accession that had the highest efficiencies in callus and green shoot formation, and we observed that light during callus culturing significantly inhibited calli growth, but promoted plant regeneration from calli in the selected accession.     

Genome-wide identification and expression analysis of the molecular chaperone binding protein <Emphasis Type="Italic">BiP</Emphasis> genes in <Emphasis Type="Italic">Citrus</Emphasis>

Here, we report for the first time the genome-wide identification and expression analysis of the molecular chaperone BiP genes in Citrus. Six genes encoding the conserved protein domain family GPR78/BiP/KAR2 were identified in the genome of Citrus sinensis and C. clementina. Two of them, named here as CsBiP1 and CsBiP2, were classified as true BiPs based on their deduced amino acid sequences. Alignment of the deduced amino acid sequences of CsBiP1 and CsBiP2 with BiP homologs from soybean and Arabidopsis showed that they contain all the conserved functional motifs of BiPs. Analysis of the promoter region of CsBiPs revealed the existence of cis-acting regulatory sequences involved in abiotic, heat-shock, and endoplasmic reticulum (ER) stress responses. Publicly available RNA-seq data indicated that CsBiP1 is abundantly expressed in leaf, flower, fruit, and callus, whereas CsBiP2 expression is rarely detected in any tissues under normal conditions. Comparative quantitative real-time PCR (qPCR) analysis of expression of these genes between C. sinensis grafted on the drought-tolerant “Rangpur” lime (C. limonia) and -sensitive “Flying Dragon” trifoliate orange (Poncirus trifoliata) rootstocks showed that CsBiP1 was upregulated by drought stress on the former but downregulated on the latter, whereas the CsBiP2 mRNA levels were downregulated on drought-stressed “Flying Dragon,” but remained constant on “Rangpur.” CsBiP2 upregulation was only observed in C. sinensis seedlings subjected to osmotic and cold treatments. Taken together, these results indicate the existence of two highly conserved BiP genes in Citrus that are differentially regulated in the different tissues and in response to abiotic stresses.     

Molecular evidence for a natural diploid hybrid between <Emphasis Type="Italic">Miscanthus</Emphasis><Emphasis Type="Italic">sinensis</Emphasis> (Poaceae) and <Emphasis Type="Italic">M. sacchariflorus</Emphasis>

The hybrid origin of Miscanthus purpurascens has previously been proposed, primarily because of its intermediate morphology. In this study, phylogenies based on the DNA sequences from the internal transcribed spacer region of nuclear ribosomal DNA (nrDNA ITS), on the DNA sequences of the trnL intron and trnL-F intergenic spacer of chloroplast DNA, and on amplified fragment length polymorphism (AFLP) fingerprinting confirm that M. purpurascens originated through homoploid hybridization between M. sinensis and M. sacchariflorus. Two different types of ITS sequences were identified from almost all plants of M. purpurascens. One type was found to be closely related to M. sinensis and the other to M. sacchariflorus. Miscanthus purpurascens was found to possess many M. sinensis- and M. sacchariflorus-specific AFLP bands but no band specific to itself. Clustering with the Unweighted Pair Group Method with Arithmetic Mean and principal coordinate analysis based on the AFLP data also demonstrated that M. purpurascens is an approximate intermediate of the two species. In addition, M. purpurascens has the plastid genome of M. sinensis or M. sacchariflorus, suggesting that either species could be its maternal parent. All specimens of M. purpurascens and its coexisting parental species are identified as diploids (2n = 2x = 38). Possible mechanisms of natural hybridization, hybrid status, chloroplast DNA recombination, and evolutionary implications of this hybridization are also discussed.     

Post-release evaluation of non-target effects of <Emphasis Type="Italic">Torymus sinensis</Emphasis>, the biological control agent of <Emphasis Type="Italic">Dryocosmus kuriphilus</Emphasis> in Italy

A post-release study was performed to assess the impact of Torymus sinensis (Hymenoptera: Torymidae), the biological control agent of Dryocosmus kuriphilus (Hymenoptera: Cynipidae), on native cynipid gall inducers in Italy. In total, 14,512 non-target galls were collected, corresponding to seven genera: Andricus, Aphelonyx, Biorhiza, Cynips, Diplolepis, Neuroterus, and Synophrus, and 8708 chalcid parasitoids were recorded. The Torymidae family accounted for about 30%, and Bootanomyia (=Megastigmus) dorsalis, Torymus affinis and T. flavipes were the most represented species. A total of 116 T. sinensis emerged from 15 different oak galls, mainly Andricus curvator and A. inflator. In controlled conditions, oviposition was recorded on A. cydoniae, A. grossulariae and A. lucidus, while no mating with native congeneric species occurred. This paper confirms the realised host-range expansion by T. sinensis. Even if it were extremely difficult to evaluate its magnitude, the impact appears minimal, and an occasional feeding with no changes in the distribution or abundance of non-target hosts is expected.     

Distribution and stress tolerance of <Emphasis Type="Italic">Fimbristylis dichotoma</Emphasis> subsp. <Emphasis Type="Italic">podocarpa</Emphasis> (Cyperaceae) growing in highly acidic solfatara fields

Solfatara fields, areas surrounding fumaroles (volcanic vents) near the hot springs or volcanoes, are characterized by severe environmental conditions such as low soil pH and high aluminum contents. Fimbristylis dichotoma subsp. podocarpa is an endangered plant distributed in solfatara fields of Kyushu, western Japan. This species is known to form stands in sites closed to fumaroles where another solfatara plant Miscanthus sinensis do not colonize. We conducted field survey and culture experiments to test the hypothesis that F. dichotoma has higher tolerance to low pH and high aluminum conditions than M. sinensis, which corresponds to the distribution pattern of the two species. In the study site of Myoban Hot Spring, Oita Prefecture, rhizosphere soils of F. dichotoma showed lower pH and higher aluminum contents than those of M. sinensis. The culture experiment showed that germination of F. dichotoma was not inhibited even at pH 2, whereas significant decline in germination (%) was observed in M. sinensis. However, because of the low germination (%) of F. dichotoma, the values of the two species were similar at pH 2. In the pot culture, seedling growth of M. sinensis was suppressed at the aluminum concentrations tested (400 mg AlCl₃ L^?1). Conversely, no significant decline in the seedling growth was observed in F. dichotoma at the same aluminum levels. Considering soil conditions in the field, we concluded that that the difference in the tolerance to aluminum between the two species played a significant role in determining their distribution pattern.     

LTR retrotransposons from the <Emphasis Type="Italic">Citrus x clementina</Emphasis> genome: characterization and application

Long terminal repeat retrotransposons (LTR-RTs) are a large portion of most plant genomes, and can be used as a powerful molecular marker system. The first citrus reference genome (Citrus x clementina) has been publicly available since 2011; however, previous studies in citrus have not utilized the whole genome for LTR-RT marker development. In this study, 3959 full-length LTR-RTs were identified in the C. x clementina genome using structure-based (LTR_FINDER) and homology-based (RepeatMasker) methods. LTR-RTs were first classified by protein domain into Gypsy and Copia superfamilies, and then clustered into 1074 families based on LTR sequence similarity. Three hundred fifty Copia families were grouped into four lineages: Retrofit, Tork, Sire, and Oryco. One hundred seventy-eight Gypsy families were sorted into six lineages: Athila, Tat, Renia, CRM, Galadriel, and Del. Most LTR-RTs (3218 or 81.3%) were anchored to the nine Clementine mandarin linkage groups, accounting for 9.74% of chromosomes currently assembled. Accessions of 25 Rutaceae species were genotyped using 17 inter-retrotransposon amplified polymorphism (IRAP) markers developed from conserved LTR regions. Sequence-specific amplified polymorphism (SSAP) makers were used to distinguish ‘Valencia’ and ‘Pineapple’ sweet oranges (C. x sinensis), and 24 sweet orange clones. LTR-RT markers developed from the Clementine genome can be transferred within the Rutaceae family demonstrating that they are an excellent tool for citrus and Rutaceae genetic analysis.