Distribution of <Emphasis Type="Italic">Divo</Emphasis> in <Emphasis Type="Italic">Coffea</Emphasis> genomes,a poorly described family of angiosperm LTR-Retrotransposons

Coffea arabica (the Arabica coffee) is an allotetraploid species originating from a recent hybridization between two diploid species: C. canephora and C. eugenioides. Transposable elements can drive structural and functional variation during the process of hybridization and allopolyploid formation in plants. To learn more about the evolution of the C. arabica genome, we characterized and studied a new Copia LTR-Retrotransposon (LTR-RT) family in diploid and allotetraploid Coffea genomes called Divo. It is a complete and relatively compact LTR-RT element (~5 kb), carrying typical Gag and Pol Copia type domains. Reverse Trancriptase (RT) domain-based phylogeny demonstrated that Divo is a new and well-supported family in the Bianca lineage, but strictly restricted to dicotyledonous species. In C. canephora, Divo is expressed and showed a genomic distribution along gene rich and gene poor regions. The copy number, the molecular estimation of insertion time and the analysis at orthologous locations of insertions in diploid and allotetraploid coffee genomes suggest that Divo underwent a different and recent transposition activity in C. arabica and C. canephora when compared to C. eugenioides. The analysis of this novel LTR-RT family represents an important step toward uncovering the genome structure and evolution of C. arabica allotetraploid genome.     

Comparative genome-wide analysis of repetitive DNA in the genus <Emphasis Type="Italic">Populus</Emphasis> L.

Genome skimming was performed, using Illumina sequence reads, in order to obtain a detailed comparative picture of the repetitive component of the genome of Populus species. Read sets of seven Populus and two Salix species (as outgroups) were subjected to clustering using RepeatExplorer (Novák et al. BMC Bioinformatics 11:378 2010). The repetitive portion of the genome ranged from 33.8 in Populus nigra to 46.5% in Populus tremuloides. The large majority of repetitive sequences were long terminal repeat-retrotransposons. Gypsy elements were over-represented compared to Copia ones, with a mean ratio Gypsy to Copia of 6.7:1. Satellite DNAs showed a mean genome proportion of 2.2%. DNA transposons and ribosomal DNA showed genome proportions of 1.8 and 1.9%, respectively. The other repeat types accounted for less of 1% each. Long terminal repeat-retrotransposons were further characterized, identifying the lineage to which they belong and studying the proliferation times of each lineage in the different species. The most abundant lineage was Athila, which showed large differences among species. Concerning Copia lineages, similar transpositional profiles were observed among all the analysed species; by contrast, differences in transpositional peaks of Gypsy lineages were found. The genome proportions of repeats were compared in the seven species, and a phylogenetic tree was built, showing species separation according to the botanical section to which the species belongs, although significant differences could be found within sections, possibly related to the different geographical origin of the species. Overall, the data indicate that the repetitive component of the genome in the poplar genus is still rapidly evolving.     

QTL mapping of mandarin (<Emphasis Type="Italic">Citrus reticulata</Emphasis>) fruit characters using high-throughput SNP markers

Seedlessness, flavor, and color are top priorities for mandarin (Citrus reticulata Blanco) cultivar improvement. Given long juvenility, large tree size, and high breeding cost, marker-assisted selection (MAS) may be an expeditious and economical approach to these challenges. The objectives of this study were to construct high-density mandarin genetic maps and to identify single nucleotide polymorphism (SNP) markers associated with fruit quality traits. Two parental genetic maps were constructed from an F₁ population derived from ‘Fortune’ × ‘Murcott’, two mandarin cultivars with distinct fruit characters, using a 1536-SNP Illumina GoldenGate assay. The map for ‘Fortune’ (FOR) consisted of 189 SNPs spanning 681.07 cM and for ‘Murcott’ (MUR) consisted of 106 SNPs spanning 395.25 cM. Alignment of the SNP sequences to the Clementine (Citrus clementina) genome showed highly conserved synteny between the genetic maps and the genome. A total of 48 fruit quality quantitative trait loci (QTLs) were identified, and ten of them stable over two or more samplings were considered as major QTLs. A cluster of QTLs for flavedo color space values L, a, b, and a/b and juice color space values a and a/b were detected in a single genomic region on linkage group 4. Two carotenoid biosynthetic pathway genes, pds1 and ccd4, were found within this QTL interval. Several SNPs were potentially useful in MAS for these fruit characteristics. QTLs were validated in 13 citrus selections, which may be useful in further validation and tentative MAS in mandarin fruit quality improvement.     

Phylogenetic relationships of Aurantioideae (Rutaceae) based on RAD-Seq

The economically and nutritionally important genus Citrus belongs to the subfamily Aurantioideae in the family Rutaceae. Here, we analyzed the phylogenetic relationships of the subfamily Aurantioideae based on RAD-Seq. The RAD-Seq data produced phylogenetic trees with high support values, clear discriminations based on branch length, and elucidations of early branching events. Our genetic classification corresponded well with the classical morphological classification system and supported the subdivision of Citreae, one of two tribes of the Aurantioideae, into three subtribes—Triphasiinae, Citrinae, and Balsamocitrinae. Additionally, it was largely consistent with the subdivision of Clauseneae, the other tribe of the Aurantioideae, into three subtribes—Micromelinae, Clauseninae, and Merrillinae; the exception was Murraya paniculata. With the exception of members of primitive citrus fruit trees, namely, Severinia buxifolia and Hesperethusa crenulata, lower-level morphological groupings under subtribes based on genetic and morphological classifications corresponded well. The phylogenetic relationship between Asian “true citrus fruit trees” (genera Citrus, Poncirus, and Fortunella) and Australian/New Guinean citrus fruit trees (genera Microcitrus, Eremocitrus, and Clymenia) was inconsistent between present classification based mainly on the nuclear genome and the previous classification based on the chloroplast genome. This inconsistency may be explained by chloroplast capture. Our findings provide a valuable insight into the genetic relationships of the subfamily Aurantioideae in the family Rutaceae.     

Long terminal repeat retrotransposons of <Emphasis Type="Italic">Mus musculus</Emphasis>

Background

Long terminal repeat (LTR) retrotransposons make up a large fraction of the typical mammalian genome. They comprise about 8% of the human genome and approximately 10% of the mouse genome. On account of their abundance, LTR retrotransposons are believed to hold major significance for genome structure and function. Recent advances in genome sequencing of a variety of model organisms has provided an unprecedented opportunity to evaluate better the diversity of LTR retrotransposons resident in eukaryotic genomes.

Results

Using a new data-mining program, LTR_STRUC, in conjunction with conventional techniques, we have mined the GenBank mouse (Mus musculus) database and the more complete Ensembl mouse dataset for LTR retrotransposons. We report here that the M. musculus genome contains at least 21 separate families of LTR retrotransposons; 13 of these families are described here for the first time.

Conclusions

All families of mouse LTR retrotransposons are members of the gypsy-like superfamily of retroviral-like elements. Several different families of unrelated non-autonomous elements were identified, suggesting that the evolution of non-autonomy may be a common event. High sequence similarity between several LTR retrotransposons identified in this study and those found in distantly-related species suggests that horizontal transfer has been a significant factor in the evolution of mouse LTR retrotransposons.

     

Structural organization of fatty acid desaturase loci in linseed lines with contrasting linolenic acid contents

Flax (Linum usitatissimum L.), the richest crop source of omega-3 fatty acids (FAs), is a diploid plant with an estimated genome size of ~370 Mb and is well suited for studying genomic organization of agronomically important traits. In this study, 12 bacterial artificial chromosome clones harbouring the six FA desaturase loci sad1, sad2, fad2a, fad2b, fad3a and fad3b from the conventional variety CDC Bethune and the high linolenic acid line M5791 were sequenced, analysed and compared to determine the structural organization of these loci and to gain insights into the genetic mechanisms underlying FA composition in flax. With one gene every 3.2–4.6 kb, the desaturase loci have a higher gene density than the genome’s average of one gene per 7.8–8.2 kb. The gene order and orientation across the two genotypes were generally conserved with the exception of the sad1 locus that was predicted to have additional genes in CDC Bethune. High sequence conservation in both genic and intergenic regions of the sad and fad2b loci contrasted with the significant level of variation of the fad2a and fad3 loci, with SNPs being the most frequently observed mutation type. The fad2a locus had 297 SNPs and 36 indels over ~95 kb contrasting with the fad2b locus that had a mere seven SNPs and four indels in ~110 kb. Annotation of the gene-rich loci revealed other genes of known role in lipid or carbohydrate metabolic/catabolic pathways. The organization of the fad2b locus was particularly complex with seven copies of the fad2b gene in both genotypes. The presence of Gypsy, Copia, MITE, Mutator, hAT and other novel repeat elements at the desaturase loci was similar to that of the whole genome. This structural genomic analysis provided some insights into the genomic organization and composition of the main desaturase loci of linseed and of their complex evolution through both tandem and whole genome duplications.     

LTR retrotransposons in the <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> and <Emphasis Type="Italic">A. nidulans</Emphasis> genomes

Fungi of the genus Aspergillus can infect all tissues and organs, causing invasive mycosis (aspergillosis). This disease can be fatal, especially in immunocompromised patients. Microbiological monitoring of these infectious agents is obligatory in modern medical facilities. Mobile elements can be used as markers to identify the Aspergillus species and strains found indoors as well as to diagnose aspergillosis. Genomic sequences of two Aspergillus species, A. fumigatus and A. nidulans, were analyzed in silico in order to detect LTR retrotransposons. These species were found to considerably differ in the composition of retrotransposon families. One of the families, present in both Aspergillus species, was phylogenetically quite different from all known fungal retrotransposons. The majority of its elements were damaged copies. Nevertheless, allegedly undamaged LTR retrotransposon copies were described that contained intact ORFs and might be active.     

Transgenic citrus expressing synthesized <Emphasis Type="Italic">cecropin</Emphasis> B genes in the phloem exhibits decreased susceptibility to Huanglongbing

Key message

Expression of synthesized cecropin B genes in the citrus phloem, where Candidatus Liberibacter asiaticus resides, significantly decreased host susceptibility to Huanglongbing.

Abstract

Huanglongbing (HLB), associated with Candidatus Liberibacter asiaticus bacteria, is the most destructive disease of citrus worldwide. All of the commercial sweet orange cultivars lack resistance to this disease. The cationic lytic peptide cecropin B, isolated from the Chinese tasar moth (Antheraea pernyi), has been shown to effectively eliminate bacteria. In this study, we demonstrated that transgenic citrus (Citrus sinensis Osbeck) expressing the cecropin B gene specifically in the phloem had a decreased susceptibility to HLB. Three plant codon-optimized synthetic cecropin B genes, which were designed to secrete the cecropin B peptide into three specific sites, the extracellular space, the cytoplasm, and the endoplasmic reticulum, were constructed. Under the control of the selected phloem-specific promoter GRP1.8, these constructs were transferred into the citrus genome. All of the cecropin B genes were efficiently expressed in the phloem of transgenic plants. Over more than a year of evaluation, the transgenic lines exhibited reduced disease severity. Bacterial populations in transgenic lines were significantly lower than in the controls. Two lines, in which bacterial populations were significantly lower than in others, showed no visible symptoms. Thus, we demonstrated the potential application of the phloem-specific expression of an antimicrobial peptide gene to protect citrus plants from HLB.

     

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.     

The complete chloroplast genome sequence of <Emphasis Type="Italic">Pseudoroegneria libanotica</Emphasis>, genomic features,and phylogenetic relationship with <Emphasis Type="Italic">Triticeae</Emphasis> species

Pseudoroegneria libanotica is an important herbage diploid species possessing the St genome. The St genome participates in the formation of nine perennial genera in Triticeae (Poaceae). The whole chloroplast (cp) genome of P. libanotica is 135 026 bp in length. The typical quadripartite structure consists of one large single copy of 80 634 bp, one small single copy of 12 766 bp and a pair of inverted regions (20 813 bp each). The cp genome contains 76 coding genes, four ribosomal RNA and 30 transfer RNA genes. Comparative sequence analysis suggested that: 1) the 737 bp deletion in the cp of P. libanotica was specific in Triticeae species and might transfer into its nuclear genome; 2) hot-spot regions, indels in intergenic regions and protein coding sequences mainly led to the length variation in Triticeae; 3) highly divergence regions combined with negative selection in rpl2, rps12, ccsA, rps8, ndhH, petD, ndhK, psbM, rps3, rps18, and ndhA were identified as effective molecular markers and could be considered in future phylogenetic studies of Triticeae species; and 4) ycf3 gene with rich cpSSRs was suitable for phylogeny analysis or could be used for DNA barcoding at low taxonomic levels. The cpSSRs distribution in the coding regions of diploid Triticeae species was shown for the first time and provided a valuable source for developing primers to study specific simple sequence repeat loci.     

Retrotransposons in <Emphasis Type="Italic">Betula nana</Emphasis>, and interspecific relationships in the Betuloideae,based on inter-retrotransposon amplified polymorphism (IRAP) markers

The Betulaceae family comprises two subfamilies, Betuloideae and Corylaceae. The subfamily Betuloideae contains two genera, Alnus Mill. and Betula L. Twenty putative long terminal repeat (LTR) retrotransposons were mined from 171 scaffolds containing 5,208,995 bp of dwarf birch (Betula nana) genome sequences. Five retrotransposons were finally selected after filtering the retrotransposon canonical features and nucleotide similarities between left and right LTR sequences. Of the five retroelements, three elements were found to be Ty1/Copia retrotransposons; identity of the other two elements could not be ascertained due to sequence undetermined ‘N’ bases in the sequence database. Inter-retrotranposon amplified polymorphism (IRAP) analysis, based on the LTR sequences of the mined LTR-retrotransposons, produced 179 discernible IRAP bands among the Alnus and Betula genera. Sequence analysis revealed no size homoplasy among the homologous IRAP bands. Phylogenetic and principle coordinate analysis, based on the band sharing among the taxa, showed the species in two different genera were clearly separated. The subgenera in each genus of Alnus and Betula were also distinguishable from the IRAP profiles. In the genus Betula, the species in subgenus Betula showed mixed clustering between species. This is incongruent with the phylogeographical distribution of the species.     

Curry leaf smells better than citrus to females of <Emphasis Type="Italic">Diaphorina citri</Emphasis> (Hemiptera: Liviidae)

The Asian citrus psyllid (ACP) Diaphorina citri Kuwayama has a host range of about 20 species of the family Rutaceae, including Citrus spp. However, few studies have reported on its host preference. This study evaluated the host-choice behavior of ACP in curry leaf (Murraya koenigii L.), through free-choice test and bioassays with a type ‘Y’ olfactometer, and also characterized the volatiles involved in attracting the ACP. In the free-choice test, the number of adults per plant on curry leaf was higher than the number on citrus plants. When the ACP was tested in the olfactometer, the females showed preference for curry leaf over citrus plants. Sixteen volatile compounds were identified in citrus and curry leaves. Qualitative and quantitative differences in the compounds released by citrus and curry leaves were determined. The volatiles present in these hosts may play an important role in the attraction of D. citri. With this information, further studies should be done to develop new management strategies for the ACP.     

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.     

Genome characteristics of the proteorhodopsin-containing marine flavobacterium <Emphasis Type="Italic">Polaribacter dokdonensis</Emphasis> DSW-5

Bacteria in the genus Polaribacter, belonging to the family Flavobacteriaceae, are typically isolated from marine environments. Polaribacter dokdonensis DSW-5, the type strain of the species, is a Gram-negative bacterium isolated from the East Sea of Korea. Whole genome shotgun sequencing was performed with the HiSeq 2000 platform and paired-end reads were generated at 188-fold coverage. The sequencing reads were assembled into two contigs with a total length of 3.08 Mb. The genome sequences of DSW-5 contain 2,776 proteincoding sequences and 41 RNA genes. Comparison of average nucleotide identities among six available Polaribacteria genomes including DSW-5 suggested that the DSW-5 genome is most similar to that of Polaribacter sp. MED152, which is a proteorhodopsin-containing marine bacterium. A phylogenomic analysis of the six Polaribacter strains and 245 Flavobacteriaceae bacteria confirmed a close relationship of the genus Polaribacter with Tenacibaculum and Kordia. DSW-5’s genome has a gene encoding proteorhodopsin and genes encoding 85 enzymes belonging to carbohydrate-active enzyme families and involved in polysaccharide degradation, which may play important roles in energy metabolism of the bacterium in the marine ecosystem. With genes for 238 CAZymes and 203 peptidases, DSW-5 has a relatively high number of degrading enzymes for its genome size suggesting its characteristics as a free-living marine heterotroph.     

Comparative analysis of complete chloroplast genome sequences of two subtropical trees, <Emphasis Type="Italic">Phoebe sheareri</Emphasis> and <Emphasis Type="Italic">Phoebe omeiensis</Emphasis> (Lauraceae)

Phoebe is an economically important genus from the family Lauraceae. It is widely distributed in tropical and subtropical Asia, but systematics of the genus is unclear, and currently there is no species-level phylogeny. Here, we determined the complete chloroplast genome sequences of two species with long-range PCR and next genome sequencing technologies, and identified mutation sites and highly variable regions. These highly variable sites were used to reconstruct the phylogeny. The plastomes of Phoebe sheareri and P. omeiensis were 152, 876, and 152, 855 bp, respectively. Comparative genomic analysis indicated that there are 222 mutation sites including 146 substitutions, 73 indels, and 3 microinversions in both plastomes. Fifty-six single-nucleotide changes were identified in gene-coding regions, and 45 microsatellite sites were found for use in species identification. Fourteen divergence hotspots of 38 variable regions were located. Phylogeny was reconstructed using a Bayesian and maximum likelihood approach for 12 Phoebe species and other five related Lauraceae based on 15 of the highly variable regions including accD-psaI, atpB-rbcL, ndhC-trnV, ndhF-rpl32, petA-psbJ, psaA, psbA-trnH, rbcL, rps8-rpl14, rps16-trnQ, rpl32-trnL, trnC-petN, trnL-trnF, trnS-trnG, and ycf1 indicated that variability in the chloroplast regions proposed as variable is enough to detect divergence events among 12 taxa of Phoebe, and that maybe also useful to help to elucidate further relationships among other taxa of the genus.     

The mitochondrial DNA of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>: complete sequence,gene content and genome organization

We present an overview of the gene content and organization of the mitochondrial genome of Dictyostelium discoideum. The mitochondria genome consists of 55,564?bp with an A + T content of 72.6%. The identified genes include those for two ribosomal RNAs (rnl and rns), 18 tRNAs, ten subunits of the NADH dehydrogenase complex (nad1, 2, 3, 4, 4L, 5, 6, 7, 9 and 11), apocytochrome b (cytb), three subunits of the cytochrome oxidase (cox1/2 and 3), four subunits of the ATP synthase complex (atp1, 6, 8 and 9), 15 ribosomal proteins, and five other ORFs, excluding intronic ORFs. Notable features of D. discoideum mtDNA include the following. (1) All genes are encoded on the same strand of the DNA and a universal genetic code is used. (2) The cox1 gene has no termination codon and is fused to the downstream cox2 gene. The 13 genes for ribosomal proteins and four ORF genes form a cluster 15.4?kb long with several gene overlaps. (3) The number of tRNAs encoded in the genome is not sufficient to support the synthesis of mitochondrial protein. (4) In total, five group I introns reside in rnl and cox1/2, and three of those in cox1/2 contain four free-standing ORFs. We compare the genome to other sequenced mitochondrial genomes, particularly that of Acanthamoeba castellanii.     

Genome-wide repeat dynamics reflect phylogenetic distance in closely related allotetraploid <Emphasis Type="Italic">Nicotiana</Emphasis> (Solanaceae)

Nicotiana sect. Repandae is a group of four allotetraploid species originating from a single allopolyploidisation event approximately 5 million years ago. Previous phylogenetic analyses support the hypothesis of N. nudicaulis as sister to the other three species. This is concordant with changes in genome size, separating those with genome downsizing (N. nudicaulis) from those with genome upsizing (N. repanda, N. nesophila, N. stocktonii). However, a recent analysis reflecting genome dynamics of different transposable element families reconstructed greater similarity between N. nudicaulis and the Revillagigedo Island taxa (N. nesophila and N. stocktonii), thereby placing N. repanda as sister to the rest of the group. This could reflect a different phylogenetic hypothesis or the unique evolutionary history of these particular elements. Here we re-examine relationships in this group and investigate genome-wide patterns in repetitive DNA, utilising high-throughput sequencing and a genome skimming approach. Repetitive DNA clusters provide support for N. nudicaulis as sister to the rest of the section, with N. repanda sister to the two Revillagigedo Island species. Clade-specific patterns in the occurrence and abundance of particular repeats confirm the original (N. nudicaulis (N. repanda (N. nesophila + N. stocktonii))) hypothesis. Furthermore, overall repeat dynamics in the island species N. nesophila and N. stocktonii confirm their similarity to N. repanda and the distinctive patterns between these three species and N. nudicaulis. Together these results suggest that broad-scale repeat dynamics do in fact reflect evolutionary history and could be predicted based on phylogenetic distance.