A high‐quality genome of taro (Colocasia esculenta (L.) Schott), one of the world's oldest crops

Taro (Colocasia esculenta (L.), Schott), from the Araceae family, is one of the oldest crops with important edible, medicinal, nutritional and economic value. Taro is a highly polymorphic species including diverse genotypes adapted to a broad range of environments, but the taro genome has rarely been investigated. Here, a high‐quality chromosome‐level genome of C. esculenta was assembled using data sequenced by Illumina, PacBio and Nanopore platforms. The assembled genome size was 2,405 Mb with a contig N50 of 400.0 kb and a scaffold N50 of 159.4 Mb. In total, 2,311 Mb (96.09%) of the contig sequences was anchored onto 14 chromosomes to form pseudomolecules, and 2,126 Mb (88.43%) was annotated as repetitive sequences. Of the 28,695 predicted protein‐coding genes, 26,215 genes (91.4%) could be functionally annotated. On the basis of phylogenetic analysis using 769 genes, C. esculenta and Spirodela polyrhiza were placed on one branch of the tree that diverged approximately 73.23 million years ago. The synteny analyses showed that there have been two whole‐genome duplication events in C. esculenta separated by a relatively short gap. According to comparative genome analysis, a larger number (1,189) of distinct gene families and long terminal repeats were enriched in C. esculenta. Our high‐quality taro genome will provide valuable resources for further genetic, ecological and evolutionary analyses of taro or other species in the Araceae.     

Development of chloroplast genomic resources for Cynara

In this study, new chloroplast (cp) resources were developed for the genus Cynara, using whole cp genomes from 20 genotypes, by means of high‐throughput sequencing technologies. Our target species included seven globe artichokes, two cultivated cardoons, eight wild artichokes, and three other wild Cynara species (C. baetica, C. cornigera and C. syriaca). One complete cp genome was isolated using short reads from a whole‐genome sequencing project, while the others were obtained by means of long‐range PCR, for which primer pairs are provided here. A de novo assembly strategy combined with a reference‐based assembly allowed us to reconstruct each cp genome. Comparative analyses among the newly sequenced genotypes and two additional Cynara cp genomes (‘Brindisino’ artichoke and C. humilis) retrieved from public databases revealed 126 parsimony informative characters and 258 singletons in Cynara, for a total of 384 variable characters. Thirty‐nine SSR loci and 34 other INDEL events were detected. After data analysis, 37 primer pairs for SSR amplification were designed, and these molecular markers were subsequently validated in our Cynara genotypes. Phylogenetic analysis based on all cp variable characters provided the best resolution when compared to what was observed using only parsimony informative characters, or only short ‘variable’ cp regions. The evaluation of the molecular resources obtained from this study led us to support the ‘super‐barcode’ theory and consider the total cp sequence of Cynara as a reliable and valuable molecular marker for exploring species diversity and examining variation below the species level.     

Callus Growth and Plantlet Regeneration in Taro, Colocasia esculenta var. esculenta (L) Schott (Araceae)

Explants of taro cultivars belonging to Colocasia esculentavar. esculenta have been nearly impossible to culture untilrecently. Here, we describe a method which induces callus formationfrom bud explants of Colocasia esculenta var. esculenta cv.Akalomamale, brings about shoot and root production, and leadsto plantlet regeneration. The medium used is half-strength Murashige-Skoog(HMS) solution containing 25 ml taro tuber extract (TE), 2 mg2, 4, 5-trichlorophenoxyacetic acid and 200 mg glutamine 1^–1.TE is an important requirement for bud explants and callus tissues.Root induction on callus-derived shoots (i.e. plantlet formation)occurs on HMS containing only 25 ml TE and 100 ml coconut water1^–1. Taro, Colocasia esculenta var. esculenta (L) Schott (Araceae), coconut water, micropropagation, plantlet regeneration, root formation, taro extract, tissue culture     

Flip‐flop organization in the chloroplast genome of Capsosiphon fulvescens (Ulvophyceae,Chlorophyta)

To better understand organelle genome evolution of the ulvophycean green alga Capsosiphon fulvescens, we sequenced and characterized its complete chloroplast genome. The circular chloroplast genome was 111,561 bp in length with 31.3% GC content that contained 108 genes including 77 protein‐coding genes, two copies of rRNA operons, and 27 tRNAs. In this analysis, we found the two types of isoform, called heteroplasmy, were likely caused by a flip‐flop organization. The flip‐flop mechanism may have caused structural variation and gene conversion in the chloroplast genome of C. fulvescens. In a phylogenetic analysis based on all available ulvophycean chloroplast genome data, including a new C. fulvescens genome, we found three major conflicting signals for C. fulvescens and its sister taxon Pseudoneochloris marina within 70 individual genes: (i) monophyly with Ulotrichales, (ii) monophyly with Ulvales, and (iii) monophyly with the clade of Ulotrichales and Ulvales. Although the 70‐gene concatenated phylogeny supported monophyly with Ulvales for both species, these complex phylogenetic signals of individual genes need further investigations using a data‐rich approach (i.e., organelle genome data) from broader taxon sampling.     

Amino acid sequences of ferredoxin isoproteins from Japanese taro (Colocasia esculenta Schott)

The amino acid sequences of ferredoxins (Fd A and Fd B) from Japanese taro (Colocasia esculenta Schott) were determined. They consisted of single polypeptide chains of 98 residues, and both Fds had molecular masses of 10700 and 10500, respectively. There was a 92% homology between the sequences of the isoproteins (Fd A and Fd B). These sequences were compared with those of the closely related plant Fds and their phylogenetic tree was constructed. Two ferredoxin isoproteins from Hawaiian taro (Colocasia esculenta Schott) were also isolated and their N-terminal sequences were determined to be identical to those of Japanese taro.     

A rare case of plastid protein‐coding gene duplication in the chloroplast genome of Euglena archaeoplastidiata (Euglenophyta)

Gene duplication is an important evolutionary process that allows duplicate functions to diverge, or, in some cases, allows for new functional gains. However, in contrast to the nuclear genome, gene duplications within the chloroplast are extremely rare. Here, we present the chloroplast genome of the photosynthetic protist Euglena archaeoplastidiata. Upon annotation, it was found that the chloroplast genome contained a novel tandem direct duplication that encoded a portion of RuBisCO large subunit (rbcL) followed by a complete copy of ribosomal protein L32 (rpl32), as well as the associated intergenic sequences. Analyses of the duplicated rpl32 were inconclusive regarding selective pressures, although it was found that substitutions in the duplicated region, all non‐synonymous, likely had a neutral functional effect. The duplicated region did not exhibit patterns consistent with previously described mechanisms for tandem direct duplications, and demonstrated an unknown mechanism of duplication. In addition, a comparison of this chloroplast genome to other previously characterized chloroplast genomes from the same family revealed characteristics that indicated E. archaeoplastidiata was probably more closely related to taxa in the genera Monomorphina, Cryptoglena, and Euglenaria than it was to other Euglena taxa. Taken together, the chloroplast genome of E. archaeoplastidiata demonstrated multiple characteristics unique to the euglenoid world, and has justified the longstanding curiosity regarding this enigmatic taxon.     

What remains from a 454 run: estimation of success rates of microsatellite loci development in selected newt species (Calotriton asper,Lissotriton helveticus,and Triturus cristatus) and comparison with Illumina‐based approaches

The development of microsatellite loci has become more efficient using next‐generation sequencing (NGS) approaches, and many studies imply that the amount of applicable loci is large. However, few studies have sought to quantify the number of loci that are retained for use out of the thousands of sequence reads initially obtained. We analyzed the success rate of microsatellite loci development for three amphibian species using a 454 NGS approach on tetra‐nucleotide motif‐enriched species‐specific libraries. The number of sequence reads obtained differed strongly between species and ranged from 19,562 for Triturus cristatus to 55,626 for Lissotriton helveticus, with 52,075 reads obtained for Calotriton asper. PHOBOS was used to identify sequences with tetra‐nucleotide repeat motifs with a minimum repeat number of ten and high quality primer binding sites. Of 107 sequences for T. cristatus, 316 for C. asper and 319 for L. helveticus, we tested the amplification success, polymorphism, and degree of heterozygosity for 41 primer combinations each for C. asper and T. cristatus, and 22 for L. helveticus. We found 11 polymorphic loci for T. cristatus, 20 loci for C. asper, and 15 loci for L. helveticus. Extrapolated, the number of potentially amplifiable loci (PALs) resulted in estimated species‐specific success rates of 0.15% (T. cristatus), 0.30% (C. asper), and 0.39% (L. helveticus). Compared with representative Illumina NGS approaches, our applied 454‐sequencing approach on specifically enriched sublibraries proved to be quite competitive in terms of success rates and number of finally applicable loci.     

Seminal Root Growth in Sorghum (Sorghum bicolor) Under Allelopathic Influences from Residues of Taro (Colocasia esculenta)

The length of the seminal root (SR) axis and the number andlength of lateral roots (LRs) of sorghum (Sorghum bicolor Moench)were markedly inhibited by taro [Colocasia esculenta (L.) Schott]residues incorporated into a sand growing medium. The sand profilewas divided equally into zones with and without residues. Productionand elongation of the first-order LRs of the SR axis facingthe zone containing taro residues were severely suppressed.On the side facing the zone that was free of residues, productionand elongation of LRs was not inhibited. SR and LR growth wasdrastically impaired and many plants were killed when taro residueswere incorporated in large amounts into the uppermost 2 cm ofthe growing medium. The activity of the allelopathic substancesin the root zone appeared to be location-specific. Sorghum bicolor, seminal root, lateral root, Colocasia esculenta, taro, taro residues, allelopathic substances, root growth     

The complete chloroplast genome of Myriophyllum spicatum reveals a 4‐kb inversion and new insights regarding plastome evolution in Haloragaceae

Myriophyllum, among the most species‐rich genera of aquatic angiosperms with ca. 68 species, is an extensively distributed hydrophyte lineage in the cosmopolitan family Haloragaceae. The chloroplast (cp) genome is useful in the study of genetic evolution, phylogenetic analysis, and molecular dating of controversial taxa. Here, we sequenced and assembled the whole chloroplast genome of Myriophyllum spicatum L. and compared it to other species in the order Saxifragales. The complete chloroplast genome sequence of M. spicatum is 158,858 bp long and displays a quadripartite structure with two inverted repeats (IR) separating the large single copy (LSC) region from the small single copy (SSC) region. Based on sequence identification and the phylogenetic analysis, a 4‐kb phylogenetically informative inversion between trnE‐trnC in Myriophyllum was determined, and we have placed this inversion on a lineage specific to Myriophyllum and its close relatives. The divergence time estimation suggested that the trnE‐trnC inversion possibly occurred between the upper Cretaceous (72.54 MYA) and middle Eocene (47.28 MYA) before the divergence of Myriophyllum from its most recent common ancestor. The unique 4‐kb inversion might be caused by an occurrence of nonrandom recombination associated with climate changes around the K‐Pg boundary, making it interesting for future evolutionary investigations.     

Development and validation of microsatellite markers for Karnal bunt (Tilletia indica) and loose smut (Ustilago segetum tritici) of wheat from related fungal species

Simple sequence repeats (SSRs) are preferred molecular markers because of their abundance, robustness, high reproducibility, high efficiency in detecting variation and suitability for high‐throughput analysis. In this study, an attempt was made to mine and analyse the SSRs from the genomes of two seed‐borne fungal pathogens, viz Ustilago maydis, which causes common smut of maize, and Tilletia horrida, the cause of rice kernel smut. After elimination of redundant sequences, 2,703 SSR loci of U. maydis were identified. Of the remaining SSRS, 44.5% accounted for di‐nucleotide repeats followed by 29.8% and 2.7% tri‐ and tetranucleotide repeats, respectively. Similarly, 2,638 SSR loci were identified in T. horrida, of which 20.2% were di‐nucleotide, 50.4% tri‐ and 20.5% tetra‐nucleotide repeats. A set of 65 SSRs designed from each fungus were validated, which yielded 23 polymorphic SSRs from Ustilago and 21 from Tilletia. These polymorphic SSR loci were also successfully cross‐amplified with the Ustilago segetum tritici and Tilletia indica. Principal coordinate analysis of SSR data clustered isolates according to their respective species. These newly developed and validated microsatellite markers may have immediate applications for detection of genetic variability and in population studies of bunt and smut of wheat and other related host plants. Moreover, this is first comprehensive report on molecular markers suitable for variability studies in wheat seed‐borne pathogens.     

Callus formation and plantlet development from axillary buds of taro

Excised lateral buds of taro [Colocasia esculenta var. esculenta (L.) A.F. Hill] developed into plantlets and formed callus if cultured on media containing taro extract. α-Naphthaleneacetic acid enhanced the process but only if taro extract was also present. The tissue requirements for this variety of taro are different from those of Colocasia esculenta var. antiquorum (L.) A.F. Hill.     

Plant regeneration in vitro of South Pacific taro (Colocasia esculenta var. esculenta cv. Akalomamale,Aracea)

Summary Axillary bud expiants from South Pacific (Solomon Islands) taro, Colocasia esculenta var. esculenta cv. Akalomamale (Araceae) cultured on a modified Murashige-Skoog medium containing 1 mg NAA 1^–1 and TE formed callus and produced multiple plantlets. Explants died if NAA was present at levels lower than 0.1 mg 1^–1. BA was not required and may have been inhibitory. Plantlets developed faster and became larger following transfer to a hormone-free medium two weeks after the start of culture. Fully grown plants were established in a potting mix and are growing well in a greenhouse.Abbreviations BA benzyladenine - BM basal medium - Ca Colocasia esculenta var. antiquorum - Ce Colocasia esculenta var. esculenta - Ck cytokinin(s) - CW coconut water - HSMSM half strength Murashige Skoog macroelements - HSMS half strength Murashige and Skoog medium - IM initial medium(ia) - MS Murashige and Skoog medium - NAA naphthaleneacetic acid - SM second medium - TE taro corm extract - UCI University of California, Irvine     

Tracing Patterns of Chloroplast Evolution in Euglenoids: Contributions from Colacium vesiculosum and Strombomonas acuminata (Euglenophyta)

The chloroplast genomes of two photosynthetic euglenoids, Colacium vesiculosum Ehrenberg (128,889 bp), and Strombomonas acuminata (Schmarda) Deflandre (144,167 bp) have been sequenced. These chloroplast genomes in combination with those of Euglena gracilis, Eutreptia viridis, and Eutreptiella gymnastica provide a snapshot of euglenoid chloroplast evolution allowing comparisons of gene content, arrangement, and expansion. The gene content of the five chloroplast genomes is very similar varying only in the presence or absence of, rrn5, roaA, psaI, psaM, rpoA, and two tRNAs. Large gene rearrangements have occurred within the C. vesiculosum and S. acuminata chloroplast genomes. Most of these rearrangements represent repositioning of entire operons rather than single genes. When compared with previously sequenced genomes, C. vesiculosum and S. acuminata chloroplast genomes more closely resemble the E. gracilis chloroplast genome in size of the genome, number of introns, and gene order than they do those of the Eutreptiales. Overall, the chloroplast genomes of these five species show an evolutionary trend toward increased intron number, a decrease in gene density, and substantial rearrangement of gene clusters.     

RECA plays a dual role in the maintenance of chloroplast genome stability in Physcomitrella patens

Chloroplast DNA (cpDNA) encodes essential genes for chloroplast functions, including photosynthesis. Homologous recombination occurs frequently in cpDNA; however, its significance and underlying mechanism remain poorly understood. In this study, we analyzed the role of a nuclear‐encoded chloroplast‐localized homolog of RecA recombinase, which is a key factor in homologous recombination in bacteria, in the moss Physcomitrella patens. Complete knockout (KO) of the P. patens chloroplast RecA homolog RECA2 caused a modest growth defect and conferred sensitivity to methyl methanesulfonate and UV. The KO mutant exhibited low recovery of cpDNA from methyl methanesulfonate damage, suggesting that RECA2 knockout impairs repair of damaged cpDNA. The RECA2 KO mutant also exhibited reduced cpDNA copy number and an elevated level of cpDNA molecule resulting from aberrant recombination between short dispersed repeats (13–63 bp), indicating that the RECA2 KO chloroplast genome was destabilized. Taken together, these data suggest a dual role for RECA2 in the maintenance of chloroplast genome stability: RECA2 suppresses aberrant recombination between short dispersed repeats and promotes repair of damaged DNA.     

Isolation and characterization of polymorphic microsatellite loci for the study of paternity and population structure in the little penguin Eudyptula minor

We isolated and characterized eight novel microsatellite loci in the little penguin Eudyptula minor, using nonradioactive polymerase chain reaction‐based techniques to screen GA and GAAA repeats from enriched genomic DNA libraries. All eight loci were polymorphic and seven were variable in our main study population (mean H_E = 0.613, mean N_A = 7.14). Cross‐amplification using a microsatellite primer developed in Spheniscus demersus (African penguin) yielded one additional polymorphic locus. This locus combined with six of the little penguin loci is suitable for paternity assignment in little penguins (exclusion probability for seven unlinked loci = 0.993).     

Evolutionary origins of taro (Colocasia esculenta) in Southeast Asia

As an ancient clonal root and leaf crop, taro (Colocasia esculenta, Araceae) is highly polymorphic with uncertain genetic and geographic origins. We explored chloroplast DNA diversity in cultivated and wild taros, and closely related wild taxa, and found cultivated taro to be polyphyletic, with tropical and temperate clades that appear to originate in Southeast Asia sensu lato. A third clade was found exclusively in wild populations from Southeast Asia to Australia and Papua New Guinea. Our findings do not support the hypothesis of taro domestication in Papua New Guinea, despite archaeological evidence for early use or cultivation there, and the presence of apparently natural wild populations in the region (Australia and Papua New Guinea).     

Genetic Variability in Taro, Colocasia esculenta (L.) Schott (Araceae)

An examination of two seedling populations indicates that geneticvariability can be obtained in taro by means of sexual propagation. Colocasia esculenta, dasheen, taro, alkaloids, anthocyanins, calcium oxalate, chlorophyll, genetic variability, nitrogen content, protein levels, in taro seedlings     

Induction of callus from axillary buds of taro (Colocasia esculenta var. esculenta,Araceae) and subsequent plantlet regeneration

Summary Axillary buds of taro (Colocasia esculenta var. esculenta, Araceae) cultured on half strength Murashige-Skoog medium (HMS) containing taro extract (HMSTE) and 2, 4, 5-trichlorophenoxyacetic acid produce a compact, hard, slow growing callus which is not very active morphogenetically and produces only a few plantlets. When cultured on HMSTE plus 5 mg 1^–1 each of naphthaleneacetic acid and benzyl adenine (HMSNB) the buds produce a fast growing, friable and morphogenetically active callus. Meristematic regions form on the friable callus after 30 days on HMSNB. If transferred to HMSTE at this point the callus gives rise to plantlets. Addition of taro extract to the media is required for the culture of buds, induction of callus and plantlet regeneration.Abbreviations BA benzyl adenine - BNA b-naphthoxyacetic acid - CW coconut water (liquid endosperm) - DW dry weight - FW fresh weight - HMS half strength Murashige-Skoog medium - HMSCW HMSTE plus 100 ml CW 1^–1 - HMSNB HMSTE plus 5 mg 1^–1 each NAA and BA - HMSTE HMS plus 25 ml taro extract 1^–1 - HMSTR HMSTE plus 2 mg 2,4,5-T 1^–1 - MNA methyl-1-naphthaleneacetate - NAA naphthaleneacetic acid - OCPAA ortho-chlorophenoxyacetic acid - TE taro extract - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

Comparative DNA sequence analyses of Pyramimonas parkeae (Prasinophyceae) chloroplast genomes

Prasinophytes form a paraphyletic assemblage of early diverging green algae, which have the potential to reveal the traits of the last common ancestor of the main two green lineages: (i) chlorophyte algae and (ii) streptophyte algae. Understanding the genetic composition of prasinophyte algae is fundamental to understanding the diversification and evolutionary processes that may have occurred in both green lineages. In this study, we sequenced the chloroplast genome of Pyramimonas parkeae NIES254 and compared it with that of P. parkeae CCMP726, the only other fully sequenced P. parkeae chloroplast genome. The results revealed that P. parkeae chloroplast genomes are surprisingly variable. The chloroplast genome of NIES254 was larger than that of CCMP726 by 3,204 bp, the NIES254 large single copy was 288 bp longer, the small single copy was 5,088 bp longer, and the IR was 1,086 bp shorter than that of CCMP726. Similarity values of the two strains were almost zero in four large hot spot regions. Finally, the strains differed in copy number for three protein‐coding genes: ycf20, psaC, and ndhE. Phylogenetic analyses using 16S and 18S rDNA and rbcL sequences resolved a clade consisting of these two P. parkeae strains and a clade consisting of these plus other Pyramimonas isolates. These results are consistent with past studies indicating that prasinophyte chloroplast genomes display a higher level of variation than is commonly found among land plants. Consequently, prasinophyte chloroplast genomes may be less useful for inferring the early history of Viridiplantae than has been the case for land plant diversification.