Atsushi Kurabayashi

In silico polymorphism analysis for the development of simple sequence repeat and transposon markers and construction of linkage map in cultivated peanut

BackgroundPeanut (Arachis hypogaea) is an autogamous allotetraploid legume (2n = 4x = 40) that is widely cultivated as a food and oil crop. More than 6,000 DNA markers have been developed in Arachis spp., but high-density linkage maps useful for genetics, genomics, and breeding have not been constructed due to extremely low genetic diversity. Polymorphic marker loci are useful for the construction of such high-density linkage maps. The present study used in silico analysis to develop simple sequence repeat-based and transposon-based markers.ResultsThe use of in silico analysis increased the efficiency of polymorphic marker development by more than 3-fold. In total, 926 (34.2%) of 2,702 markers showed polymorphisms between parental lines of the mapping population. Linkage analysis of the 926 markers along with 253 polymorphic markers selected from 4,449 published markers generated 21 linkage groups covering 2,166.4 cM with 1,114 loci. Based on the map thus produced, 23 quantitative trait loci (QTLs) for 15 agronomical traits were detected. Another linkage map with 326 loci was also constructed and revealed a relationship between the genotypes of the FAD2 genes and the ratio of oleic/linoleic acid in peanut seed.ConclusionsIn silico analysis of polymorphisms increased the efficiency of polymorphic marker development, and contributed to the construction of high-density linkage maps in cultivated peanut. The resultant maps were applicable to QTL analysis. Marker subsets and linkage maps developed in this study should be useful for genetics, genomics, and breeding in Arachis. The data are available at the Kazusa DNA Marker Database (http://marker.kazusa.or.jp).

Complete mitochondrial DNA sequence of the endangered frog Odorrana ishikawae (family Ranidae) and unexpected diversity of mt gene arrangements in ranids

We determined the complete nucleotide sequence of the mitochondrial (mt) genome of an endangered Japanese frog, Odorrana ishikawae (family Ranidae). We also sequenced partial mt genomes of three other Odorrana and six ranid species to survey the diversity of genomic organizations and elucidate the phylogenetic problems remaining in this frog family. The O. ishikawae mt genome contained the 37 mt genes and single control region (CR) typically found in vertebrate mtDNAs, but the region of Light-strand replication origin (OL) was triplicated in this species. Four protein-encoding genes (atp6, nd2, nd3, and nd5) were found to have high sequence divergence and to be usable for population genetics studies on this endangered species. Among the surveyed ranids, only two species (Rana and Lithobates) manifested the typical neobatrachian-type mt gene arrangement. In contrast, relatively large gene rearrangements were found in Amolops, Babina, and Staurois species; and translocations of single tRNA genes (trns) were observed in Glandirana and Odorrana species. Though the inter-generic and interspecific relationships of ranid taxa remain to be elucidated based on 12S and 16S rrn sequence data, some of the derived mt gene orders were found to have synapomorphic features useful for solving problematic ranid phylogenies. The tandem duplication and random loss (TDRL) model, the traditional model for mt gene rearrangement, failed to easily explain several of the mt gene rearrangements observed here. Indeed, the recent recombination-based gene rearrangement models seemed to be more suitable for this purpose. The high frequency of gene translocations involving a specific trn block (trnH-trnS1) and several single tRNA genes suggest that there may be a retrotranslocation in ranid mt genomes.

Coexpression Analysis of Tomato Genes and Experimental Verification of Coordinated Expression of Genes Found in a Functionally Enriched Coexpression Module

Gene-to-gene coexpression analysis is a powerful approach to infer the function of uncharacterized genes. Here, we report comprehensive identification of coexpression gene modules of tomato (Solanum lycopersicum) and experimental verification of coordinated expression of module member genes. On the basis of the gene-to-gene correlation coefficient calculated from 67 microarray hybridization data points, we performed a network-based analysis. This facilitated the identification of 199 coexpression modules. A gene ontology annotation search revealed that 75 out of the 199 modules are enriched with genes associated with common functional categories. To verify the coexpression relationships between module member genes, we focused on one module enriched with genes associated with the flavonoid biosynthetic pathway. A non-enzyme, non-transcription factor gene encoding a zinc finger protein in this module was overexpressed in S. lycopersicum cultivar Micro-Tom, and expression levels of flavonoid pathway genes were investigated. Flavonoid pathway genes included in the module were up-regulated in the plant overexpressing the zinc finger gene. This result demonstrates that coexpression modules, at least the ones identified in this study, represent actual transcriptional coordination between genes, and can facilitate the inference of tomato gene function.

The Genus Fejervarya (Anura: Ranidae) in Central Western Ghats, India, with Descriptions of Four New Cryptic Species

ABSTRACT We compared six taxa of the genus Fejervarya from central Western Ghats, southwestern India, including F. rufescens, F. sahyadris, and four taxa that possess distinct mtDNA haplotypes as demonstrated by our previous studies. Morphological comparisons with F. brevipalmata, F. keralensis, F. nilagirica, and F. syhadrensis on the basis of literature descriptions and museum specimens revealed that the four haplotypes do not correspond to any of the previously described species. Therefore, they are named herein as new species. Although each of these new species was separated clearly by discriminant analyses, two large-bodied species, as well as two small-bodied species, occurring sympatrically or parapatrically in many collecting sites, were very similar to each other in external appearance. Acoustic characteristics available for five of the six species were most conspicuous and diagnostic features. This study revealed the occurrence of active speciation in Fejervarya in the Western Ghats, one of the hot spots of biodiversity in the world.

PCR Primers for the Neobatrachian Mitochondrial Genome

Abstract: Neobatrachia is a phylogenetically-nested anuran group and contains approximately 85% of amphibian species. It is difficult to analyze the neobatrachian mitochondrial (mt) genome due to accelerated nucleotide substitution rates and occasional gene rearrangements. To overcome these problems, we designed 15 PCR primers. These primers, together with two primers from a previous study, could amplify almost the entire mt genome in all neobatrachians tested (seven species from seven different families) without optimizing PCR conditions. Among the tested taxa, Trichobatrachus robustus (Arthroleptidae) and Hyperolius viridiflavus (Hyperoliidae) showed unique amplification patterns, suggesting occurrences of gene duplication and/or rearrangements in these mt genomes.

Accumulation Mechanism of γ-Aminobutyric Acid in Tomatoes (Solanum lycopersicum L.) under Low O2 with and without CO2

The storage of ripe tomatoes in low-O(2) conditions with and without CO(2) promotes γ-aminobutyric acid (GABA) accumulation. The activities of glutamate decarboxylase (GAD) and α-ketoglutarate-dependent GABA transaminase (GABA-TK) were higher and lower, respectively, following storage under hypoxic (2.4 or 3.5% O(2)) or adjusted aerobic (11% O(2)) conditions compared to the activities in air for 7 days at 25 °C. GAD activity was consistent with the expression level of mRNA for GAD. The GABA concentration in tomatoes stored under hypoxic conditions and adjusted aerobic conditions was 60-90% higher than that when they are stored in air on the same day. These results demonstrate that upregulation of GAD activity and downregulation of GABA-TK activity cause GABA accumulation in tomatoes stored under low-O(2) conditions. Meanwhile, the effect of CO(2) on GABA accumulation is probably minimal.

Genetic divergence and evolutionary relationships in six species of genera Hoplobatrachus and Euphlyctis (Amphibia: Anura) from Bangladesh and other Asian countries revealed by mitochondrial gene sequences

To elucidate the species composition, genetic divergence, evolutionary relationships, and divergence time of Hoplobatrachus and Euphlyctis frogs (subfamily Dicroglossinae, family Ranidae) in Bangladesh and other Asian countries, we analyzed the mitochondrial Cyt b, 12S, and 16S rRNA genes of 252 specimens. Our phylogenetic analyses showed 13 major clades corresponding to several cryptic species as well as to nominal species in the two genera. The results suggested monophyly of Asian Hoplobatrachus species, but the position of African Hoplobatrachus occipitalis was not clarified. Nucleotide divergence and phylogenetic data suggested the presence of allopatric cryptic species allied to Euphlyctis hexadactylus in Sundarban, Bangladesh and several parapatric cryptic species in the Western Ghats, India. The presence of at least two allopatric cryptic species among diverged Euphlyctis cyanophlyctis in Bangladesh, India, and Sri Lanka was also suggested. In some cases, our estimated divergence times matched the paleogeological events of South and Southeast Asian regions that may have led to the divergence of Hoplobatrachus and Euphlyctis taxa. Especially, land formation at Bangladesh (15-10Ma) may have allowed the spread of these frog taxa to Southeast Asian areas, and the aridification of central India (5.1-1.6Ma) might have affected the gene flow of widely distributed species. The present study revealed prior underestimation of the richness of the amphibian fauna in this region, indicating the possible occurrence of many cryptic species among these groups.

Molecular Phylogeny of the Ranid Frogs from Southwest India Based on the Mitochondrial Ribosomal RNA Gene Sequences

Abstract The Western Ghats of Southwestern India are known as one of the worlds “hotspots” of biodiversity. We collected frog specimens from the family Ranidae and investigated the phylogenetic relationships among ranid species, particularly among the Fejervarya, a genus whose morphological diagnostic characteristics and phylogenetic features remain little known. We analyzed partial sequences of the mitochondrial 12S (428 bp) and 16S rDNAs (549 bp). Results showed that the members of Fejervarya form a monophyletic group with the genera Hoplobatrachus and Euphlyctis among ranid genera. This confirms the recent allocation of Fejervarya, including it within the subfamily Dicroglossinae. The mitochondrial rDNA data in our study also appeared to be useful as a marker to distinguish Fejervarya species without morphological differences. The phylogenetic tree based on the 16S rDNA sequence showed a correlation between Fejervarya phylogenies and their geographic distributions. Lastly, our results suggested the recent occurrence of a radiation event of Fejervarya species in the Indian-Sri Lankan region.

From Antarctica or Asia? New colonization scenario for Australian-New Guinean narrow mouth toads suggested from the findings on a mysterious genus Gastrophrynoides

BackgroundMicrohylidae is a geographically widespread family of anurans. Although several extensive molecular analyses have attempted to elucidate their subfamilial relationships, and correlate these with Mesozoic and Cenozoic continental drifts, consensus has not been reached. Further, generic level relationships have not been well investigated in some microhylid subfamilies, and therefore subfamilial affiliations of some genera are still unclear. To elucidate the phylogenetic positions of two mysterious Asian genera, Gastrophrynoides and Phrynella, and to better understand the trans-continental distributions of microhylid taxa, we performed molecular phylogenetic and dating analyses using the largest molecular dataset applied to these taxa to date.ResultsSix nuclear and two mitochondrial genes (approx. 8 kbp) were sequenced from 22 microhylid frog species representing eight subfamilies. The maximum likelihood and Bayesian analyses could not fully elucidate the subfamilial relationships, suggesting a rapid radiation of these taxa between 85 and 66 million years ago. In contrast, generic relationships of Asian microhylines were generally well resolved.ConclusionOur results clearly showed that one of two problematic Asian genera, Phrynella, was nested in the clade of the Asian subfamily Microhylinae. By contrast, Gastrophrynoides occupied the most basal position of the Australian-New Guinean subfamily Asterophryinae. The estimated divergence of Gastrophrynoides from other asterophryine was unexpectedly around 48 million years ago. Although a colonization scenario via Antarctica to the Australian-New Guinean landmass has been suggested for Asterophryinae, our finding suggested a novel colonization route via Indo-Eurasia.

Afrobatrachian mitochondrial genomes: genome reorganization, gene rearrangement mechanisms, and evolutionary trends of duplicated and rearranged genes

BackgroundMitochondrial genomic (mitogenomic) reorganizations are rarely found in closely-related animals, yet drastic reorganizations have been found in the Ranoides frogs. The phylogenetic relationships of the three major ranoid taxa (Natatanura, Microhylidae, and Afrobatrachia) have been problematic, and mitogenomic information for afrobatrachians has not been available. Several molecular models for mitochondrial (mt) gene rearrangements have been proposed, but observational evidence has been insufficient to evaluate them. Furthermore, evolutionary trends in rearranged mt genes have not been well understood. To gain molecular and phylogenetic insights into these issues, we analyzed the mt genomes of four afrobatrachian species (Breviceps adspersus, Hemisus marmoratus, Hyperolius marmoratus, and Trichobatrachus robustus) and performed molecular phylogenetic analyses. Furthermore we searched for two evolutionary patterns expected in the rearranged mt genes of ranoids.ResultsExtensively reorganized mt genomes having many duplicated and rearranged genes were found in three of the four afrobatrachians analyzed. In fact, Breviceps has the largest known mt genome among vertebrates. Although the kinds of duplicated and rearranged genes differed among these species, a remarkable gene rearrangement pattern of non-tandemly copied genes situated within tandemly-copied regions was commonly found. Furthermore, the existence of concerted evolution was observed between non-neighboring copies of triplicated 12S and 16S ribosomal RNA regions.ConclusionsPhylogenetic analyses based on mitogenomic data support a close relationship between Afrobatrachia and Microhylidae, with their estimated divergence 100 million years ago consistent with present-day endemism of afrobatrachians on the African continent. The afrobatrachian mt data supported the first tandem and second non-tandem duplication model for mt gene rearrangements and the recombination-based model for concerted evolution of duplicated mt regions. We also showed that specific nucleotide substitution and compositional patterns expected in duplicated and rearranged mt genes did not occur, suggesting no disadvantage in employing these genes for phylogenetic inference.