Isolation and characterization of resistance gene analogues from Psilanthus species that represent wild relatives of cultivated coffee endemic to India.

Biotic or abiotic stress can cause considerable damage to crop plants that can be managed by building disease resistance in the cultivated gene pool through breeding for disease resistance genes (R-genes). R-genes, conferring resistance to diverse pathogens or pests share a high level of similarity at the DNA and protein levels in different plant species. This property of R-genes has been successfully employed to isolate putative resistance gene analogues (RGAs) using a PCR-based approach from new plant sources. Using a similar approach, in the present study, we have successfully amplified putative RGAs having nucleotide-binding-site leucine-rich repeats (NBS-LRR-type RGAs) from seven different sources: two cultivated coffee species (Coffea arabica L. and Coffea canephora Pierre ex. A. Froehner), four related taxa endemic to India (wild tree coffee species: Psilanthus bengalensis (Roem. & Schuttles) J.-F. Leroy, Psilanthus khasiana , Psilanthus travencorensis (Wight & Arn.) J.-F. Leroy, Psilanthus weightiana (Wall. ex Wight & Arn.) J.-F. Leroy), and a cDNA pool originally prepared from light- and drought-stressed Coffea arabica L. leaves. The total PCR amplicons obtained using NBS-LRR-specific primers from each source were cloned and transformed to construct seven independent libraries, from which 434 randomly picked clones were sequenced. In silico analysis of the sequenced clones revealed 27 sequences that contained characteristic RGA motifs, of which 24 had complete uninterrupted open reading frames. Comparisons of these with published RGAs showed several of these to be novel RGA sequences. Interestingly, most of such novel RGAs belonged to the related wild Psilanthus species. The data thus suggest the potential of the secondary gene pool as possible untapped donors of resistance genes to the present day cultivated species of coffee.

Genetic heterogeneity in wild isolates of cellular slime mold social groups.

This study addresses the issues of spatial distribution, dispersal, and genetic heterogeneity in social groups of the cellular slime molds (CSMs). The CSMs are soil amoebae with an unusual life cycle that consists of alternating solitary and social phases. Because the social phase involves division of labor with what appears to be an extreme form of "altruism", the CSMs raise interesting evolutionary questions regarding the origin and maintenance of sociality. Knowledge of the genetic structure of social groups in the wild is necessary for answering these questions. We confirm that CSMs are widespread in undisturbed forest soil from South India. They are dispersed over long distances via the dung of a variety of large mammals. Consistent with this mode of dispersal, most social groups in the two species examined for detailed study, Dictyostelium giganteum and Dictyostelium purpureum, are multi-clonal.

Multiple alternative splicing of Dmrt1 during gonadogenesis in Indian mugger, a species exhibiting temperature-dependent sex determination.

Dmrt1 is an evolutionarily conserved gene having important role in the sex determination from lower vertebrates to mammals. Recent studies show transcriptional diversity for this important gene during gonadal differentiation in a few vertebrate species having genetic sex determination (GSD). In this study, we show for the first time that the transcriptional diversity of Dmrt1 is also found in the Indian mugger that exhibits temperature-dependent sex determination (TSD). We report here isolation and characterization of eight novel isoforms of Dmrt1 from Crocodylus palustris, along with its genomic locus that is referred as, cpDmrt1. Further, by sequence comparisons of cpDmrt1 and its expressed isoforms, we demonstrate that all the isoforms are generated by alternative splicing, exonization of intronic sequences and alternative polyA sites from the same locus. The eight transcripts range from 494 to 2060 bp and encode six predicted proteins having the characteristic DM domain of Dmrt1. The major heterogeneity in the isoforms and their predicted proteins is seen only in their C-termini and 3'-UTRs, which do not match with any similar sequences reported for other vertebrates. The cpDmrt1 expression was seen mainly in developing GAM (genital ridge-adrenal-mesonephros complex) with significant upregulation only in male embryos from the start of the temperature sensitive period (TSP). More significantly, approximately 70% of this expression was contributed by only one isoform (cpDmrt1e) that also has a unique 15 amino acid domain towards its C-terminal. cpDmrt1 expression was also detected at a lower level in brain and developing kidney. The study thus provides the first account of Dmrt1 locus, its transcriptional diversity and sex-specific expression in Indian mugger, a TSD species.

Development of new genomic microsatellite markers from robusta coffee (Coffea canephora Pierre ex A. Froehner) showing broad cross-species transferability and utility in genetic studies.

BACKGROUND: Species-specific microsatellite markers are desirable for genetic studies and to harness the potential of MAS-based breeding for genetic improvement. Limited availability of such markers for coffee, one of the most important beverage tree crops, warrants newer efforts to develop additional microsatellite markers that can be effectively deployed in genetic analysis and coffee improvement programs. The present study aimed to develop new coffee-specific SSR markers and validate their utility in analysis of genetic diversity, individualization, linkage mapping, and transferability for use in other related taxa. RESULTS: A small-insert partial genomic library of Coffea canephora, was probed for various SSR motifs following conventional approach of Southern hybridisation. Characterization of repeat positive clones revealed a very high abundance of DNRs (1/15 Kb) over TNRs (1/406 kb). The relative frequencies of different DNRs were found as AT >> AG > AC, whereas among TNRs, AGC was the most abundant repeat. The SSR positive sequences were used to design 58 primer pairs of which 44 pairs could be validated as single locus markers using a panel of arabica and robusta genotypes. The analysis revealed an average of 3.3 and 3.78 alleles and 0.49 and 0.62 PIC per marker for the tested arabicas and robustas, respectively. It also revealed a high cumulative PI over all the markers using both sib-based (10-6 and 10-12 for arabicas and robustas respectively) and unbiased corrected estimates (10-20 and 10-43 for arabicas and robustas respectively). The markers were tested for Hardy-Weinberg equilibrium, linkage dis-equilibrium, and were successfully used to ascertain generic diversity/affinities in the tested germplasm (cultivated as well as species). Nine markers could be mapped on robusta linkage map. Importantly, the markers showed ~92% transferability across related species/genera of coffee. CONCLUSION: The conventional approach of genomic library was successfully employed although with low efficiency to develop a set of 44 new genomic microsatellite markers of coffee. The characterization/validation of new markers demonstrated them to be highly informative, and useful for genetic studies namely, genetic diversity in coffee germplasm, individualization/bar-coding for germplasm protection, linkage mapping, taxonomic studies, and use as conserved orthologous sets across secondary genepool of coffee. Further, the relative frequency and distribution of different SSR motifs in coffee genome indicated coffee genome to be relatively poor in microsatellites compared to other plant species.

Draft Genome Sequence of a Versatile Hydrocarbon-Degrading Bacterium, Rhodococcus pyridinivorans Strain KG-16, Collected from Oil Fields in India.

We describe here a 5.8-Mb draft genome sequence of Rhodococcus pyridinivorans strain KG-16, which was obtained from the soil samples collected from the oilfields of Krishna-Godavari basin in India. This genomic resource can provide insights into the pathways and mechanisms of hydrocarbon degradation and potentially aid in bioremediation applications.

A new genus and species of arboreal toad with phytotelmonous larvae, from the Andaman Islands, India (Lissamphibia, Anura, Bufonidae).

A new bufonid amphibian, belonging to a new monotypic genus, is described from the Andaman Islands, in the Bay of Bengal, Republic of India, based on unique external morphological and skeletal characters which are compared with those of known Oriental and other relevant bufonid genera. Blythophryne gen. n. is distinguished from other bufonid genera by its small adult size (mean SVL 24.02 mm), the presence of six presacral vertebrae, an absence of coccygeal expansions, presence of an elongated pair of parotoid glands, expanded discs at digit tips and phytotelmonous tadpoles that lack oral denticles. The taxonomic and phylogenetic position of the new taxon (that we named as Blythophryne beryet gen. et sp. n.) was ascertained by comparing its 12S and 16S partial genes with those of Oriental and other relevant bufonid lineages. Resulting molecular phylogeny supports the erection of a novel monotypic genus for this lineage from the Andaman Islands of India.

Draft Genome Sequence of Hydrocarbon-Degrading Pseudomonas putida Strain KG-4, Isolated from Soil Samples Collected from Krishna-Godavari Basin in India.

We report here the 5.58-Mb draft genome of Pseudomonas putida strain KG-4 obtained from the oil fields of the Krishna-Godavari basin, Andhra Pradesh, India. The genome sequence is expected to facilitate identification and understanding of genes associated with hydrocarbon metabolism, which can help in developing strategies for managing oil spills and bioremediation.

Draft Genome Sequence of Rhodococcus rhodochrous Strain KG-21, a Soil Isolate from Oil Fields of Krishna-Godavari Basin, India.

Here, we present the 6.1-Mb draft genome sequence of Rhodococcus rhodochrous strain KG-21, a soil isolate from the oil fields of Krishna-Godavari Basin in Andhra Pradesh, India. This genomic resource may help in the identification of the gene(s) involved in hydrocarbon degradation and their possible deployment for bioremediation.

Draft Genome Sequences of Two Drug-Resistant Isolates of Pseudomonas aeruginosa Obtained from Keratitis Patients in India.

We report here the draft genomes of two drug (fluoroquinolone)-resistant clinical isolates of Pseudomonas aeruginosa obtained from the corneal scrapings of keratitis patients from India. The two annotated genomes are 6.31 Mb and 6.41 Mb in size. These genomes are expected to facilitate the identification and understanding of the genes associated with acquired multidrug resistance.

Advantages of using mitochondrial 16S rDNA sequences to classify clinical isolates of Acanthamoeba.

PURPOSE: This work was intended to test the classification of Acanthamoeba into genotypes based on nuclear ribosomal RNA gene (18S rDNA, Rns) sequences. Nearly all Acanthamoeba keratitis (AK) isolates are genotype RnsT4. This marked phylogenetic localization is presumably either due to an innate potential for pathogenicity or to a peculiarity of the gene sequences used. To differentiate between these possibilities, relationships among isolates have been reexamined, using a second gene. METHODS: Phylogenetic relationships among isolates of Acanthamoeba were studied, using sequences of the mitochondrial small subunit ribosomal RNA gene (16S rDNA; rns). Genotypes based on complete sequences of approximately 1540 bp were determined for 68 strains, by using multiple phylogenetic analyses. RESULTS: Each strain's mitochondria contained a single intron-free rns sequence (allele). The 68 strains had 35 different sequences. Twenty-eight strains had unique sequences, and 40 strains each shared one of the seven remaining sequences. Eleven mitochondrial rns genotypes corresponding to 11 of 12 previously described nuclear Rns genotypes were identified. Genotype rnsT4 was subdivided into eight distinct clades, with seven including Acanthamoeba keratitis (AK) isolates. CONCLUSIONS: The phylogenetic clustering of AK isolates was confirmed and thus is not specific to the nuclear gene. Rns and rns sequences are both suitable for genotyping of ACANTHAMOEBA: However, the mitochondrial sequences are shorter and more consistent in length, have a higher percentage of alignable bases for sequence comparisons, and have none of the complications caused by multiple alleles or introns, which are occasionally found in Rns. In addition, the more common occurrence of strains with identical rns sequences simplifies identification and clustering of isolates.

Acanthamoeba keratitis in non-contact lens wearers in India: DNA typing-based validation and a simple detection assay.

OBJECTIVES: To establish that the protozoan Acanthamoeba is one of the causative organisms associated with non-contact lens-related keratitis in the Indian population and to develop a simple and sensitive diagnostic assay for clinical testing. DESIGN: DNA sequencing of nuclear 18S and 26S ribosomal DNA motifs was performed and compared with the reference Acanthamoeba strains, to establish the genetic identity of the putative amoeba isolates obtained from the corneal scrapings of non-contact lens-wearing patients with keratitis. Ribosomal DNA typing of clinical corneal scrapings from the patients with keratitis was performed by means of a simple agarose gel-based multiplex polymerase chain reaction assay, to detect the cases of Acanthamoeba keratitis. RESULTS: The ribosomal DNA analysis of 15 putative amoeba isolates obtained from the corneal scrapings of 14 patients with keratitis and 1 from the patients' environment established the isolates to be pathogenic formsof Acanthamoeba belonging to type T4 ribosomal DNA genotype. Multiplex polymerase chain reaction assay was specific and sensitive enough to detect as low as 5 pg of Acanthamoeba DNA. Its utility as a reliable diagnostic assay was demonstrated directly with the use of 34 additional corneal scrapings. CONCLUSIONS: Acanthamoeba is one of the causative organisms of keratitis in Indian patients with no history of contact lens usage. Moreover, the Acanthamoeba infection can be easily detected in the clinical samples by means of the simple multiplex polymerase chain reaction assay based on ribosomal DNA typing. Clinical Relevance This study suggests the need and means to determine the incidence and prevalance of Acanthamoeba keratitis in India and elsewhere. Moreover, the polymerase chain reaction assay would help in early and definitive diagnosis, leading to better prognosis of Acanthamoeba keratitis condition.

Cloning and sequencing of complete tau-crystallin cDNA from embryonic lens of Crocodylus palustris.

tau-Crystallin is a taxon-specific structural protein found in eye lenses. We present here the cloning and sequencing of complete tau-crystallin cDNA from the embryonic lens of Crocodylus palustris and establish it to be identical to the a-enolase gene from non-lenticular tissues. Quantitatively, the tau-crystallin was found to be the least abundant crystallin of the crocodilian embryonic lenses. Crocodile tau-crystallin cDNA was isolated by RT-PCR using primers designed from the only other reported sequence from duck and completed by 5'- and 3'-rapid amplification of cDNA ends (RACE) using crocodile gene specific primers designed in the study. The complete tau-crystallin cDNA of crocodile comprises 1305 bp long ORF and 92 and 409 bp long untranslated 5'- and 3'-ends respectively. Further, it was found to be identical to its putative counterpart enzyme a-enolase, from brain, heart and gonad, suggesting both to be the product of the same gene. The study thus provides the first report on cDNA sequence of tau-crystallin from a reptilian species and also re-confirms it to be an example of the phenomenon of gene sharing as was demonstrated earlier in the case of peking duck. Moreover, the gene lineage reconstruction analysis helps our understanding of the evolution of crocodilians and avian species.

Development of genic and genomic SSR markers of robusta coffee (Coffea canephora Pierre Ex A. Froehner).

Coffee breeding and improvement efforts can be greatly facilitated by availability of a large repository of simple sequence repeats (SSRs) based microsatellite markers, which provides efficiency and high-resolution in genetic analyses. This study was aimed to improve SSR availability in coffee by developing new genic-/genomic-SSR markers using in-silico bioinformatics and streptavidin-biotin based enrichment approach, respectively. The expressed sequence tag (EST) based genic microsatellite markers (EST-SSRs) were developed using the publicly available dataset of 13,175 unigene ESTs, which showed a distribution of 1 SSR/3.4 kb of coffee transcriptome. Genomic SSRs, on the other hand, were developed from an SSR-enriched small-insert partial genomic library of robusta coffee. In total, 69 new SSRs (44 EST-SSRs and 25 genomic SSRs) were developed and validated as suitable genetic markers. Diversity analysis of selected coffee genotypes revealed these to be highly informative in terms of allelic diversity and PIC values, and eighteen of these markers (∼ 27%) could be mapped on a robusta linkage map. Notably, the markers described here also revealed a very high cross-species transferability. In addition to the validated markers, we have also designed primer pairs for 270 putative EST-SSRs, which are expected to provide another ca. 200 useful genetic markers considering the high success rate (88%) of marker conversion of similar pairs tested/validated in this study.

Association of leaf micro-morphological characters with powdery mildew resistance in field-grown mulberry (Morus spp.) germplasm.

BACKGROUND AND AIMS: Micro-morphological characteristics can influence fungal infectivity. We sought links between micro-morphology and resistance to powdery mildew in mulberry with the intention of assisting selection of disease-resistant lines. METHODOLOGY: Over 3 years and under field conditions, we evaluated 30 lines of mulberry with contrasting susceptibilities to powdery mildew (15 resistant and 15 susceptible). Disease severity was related statistically to stomatal area, stomatal density, stomatal index, upper and lower cuticular thicknesses, leaf thickness and trichome density. PRINCIPAL RESULTS: Differences between lines were significant (P <0.05) for all characters studied. Variation between the resistant and susceptible groups was statistically highly significant (P <0.01) for stomatal index, stomatal area and trichome density. The powdery mildew-resistant group was distinguished by  17.4 % lower stomatal density, 12.5 % smaller stomatal index per unit leaf area, 20.0 % greater trichome density and 18.0 % higher stomatal area compared with the susceptible group. Trichome density was negatively correlated with disease severity index and with the accumulative area under disease progression curves. Stomatal density was positively correlated with both measures of disease severity. Although stomatal area was negatively related to disease severity index (r = -0.28; P <0.05), the correlation was weak. There was no statistically significant relationship between stomatal area and the accumulative area under disease progression curves. The germplasm was partitioned into seven sub-groups based on hierarchical cluster analysis derived from pooled disease severity index scores and three highly significant micro-morphological characters. Eighty per cent of the resistant germplasm accumulated in three cluster components (A1, A2 and B2) characterized by high trichome densities and a high stomatal density and stomatal index. CONCLUSIONS: Resistance to powdery mildew in mulberry is associated with trichome and stomatal features rather than leaf and epidermal thicknesses. Trichome density, stomatal density and stomatal index are shown to be promising markers for screening powdery mildew resistance in breeding programmes.

Permanent genetic resources added to Molecular Ecology Resources Database 1 August 2010-30 September 2010.

This article documents the addition of 229 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Acacia auriculiformis × Acacia mangium hybrid, Alabama argillacea, Anoplopoma fimbria, Aplochiton zebra, Brevicoryne brassicae, Bruguiera gymnorhiza, Bucorvus leadbeateri, Delphacodes detecta, Tumidagena minuta, Dictyostelium giganteum, Echinogammarus berilloni, Epimedium sagittatum, Fraxinus excelsior, Labeo chrysophekadion, Oncorhynchus clarki lewisi, Paratrechina longicornis, Phaeocystis antarctica, Pinus roxburghii and Potamilus capax. These loci were cross-tested on the following species: Acacia peregrinalis, Acacia crassicarpa, Bruguiera cylindrica, Delphacodes detecta, Tumidagena minuta, Dictyostelium macrocephalum, Dictyostelium discoideum, Dictyostelium purpureum, Dictyostelium mucoroides, Dictyostelium rosarium, Polysphondylium pallidum, Epimedium brevicornum, Epimedium koreanum, Epimedium pubescens, Epimedium wushanese and Fraxinus angustifolia.

Male-specific expression of Sox9 during gonad development of crocodile and mouse is mediated by alternative splicing of its proline-glutamine-alanine rich domain.

The initial trigger for sexual differentiation is regulated by multiple ways during embryonic development. In vertebrates, chromosome-based mechanisms generally known as genetic sex determination are prevalent; however, some species, such as many reptilians, display temperature-dependent sex determination. The Sry-related transcription factor, Sox9, which is expressed by an evolutionary conserved gene, has been shown to be a key player in the process of sex determination. In the present study, we report the identification and expression of crocodile homolog of Sox9 (cpSox9) from the Indian Mugger, Crocodylus palustris. We show that cpSox9 undergoes extensive alternative splicing around the proline-glutamine-alanine rich transactivation domain that results in cpSox9 variants with presumably impaired or reduced transactivation potential. The multiple isoforms were also detected in various embryonic tissues, with some of them displaying a differential expression profile. With respect to sex differentiation, a putative unspliced full-length cpSox9 could be detected only in the genital ridge-adrenal-mesonephros complex of male, but not female embryos during the temperature-sensitive period. Importantly, we further show that this phenomenon was not restricted to the temperature-dependent sex determination species C. palustris, but was also observed in the mouse, a species exhibiting genetic sex determination. Thus, the present study describes, for the first time, a complete coding locus of Sox9 homolog from a temperature-dependent sex determination species. More importantly, we demonstrate an evolutionarily conserved role of alternative splicing resulting in transcriptional diversity and male-sex specific expression of Sox9 during testis development in vertebrates (i.e. irrespective of their underlying sex-determination mechanisms).

Extended mitogenomic phylogenetic analyses yield new insight into crocodylian evolution and their survival of the Cretaceous-Tertiary boundary.

The mitochondrial genomes of the dwarf crocodile, Osteolaemus tetraspis, and two species of dwarf caimans, the smooth-fronted caiman, Paleosuchus trigonatus, and Cuvier's dwarf caiman, Paleosuchus palpebrosus, were sequenced and included in a mitogenomic phylogenetic study. The phylogenetic analyses, which included a total of ten crocodylian species, yielded strong support to a basal split between Crocodylidae and Alligatoridae. Osteolaemus fell within the Crocodylidae as the sister group to Crocodylus. Gavialis and Tomistoma, which joined on a common branch, constituted a sister group to Crocodylus/Osteolaemus. This suggests that extant crocodylians are organized in two families: Alligatoridae and Crocodylidae. Within the Alligatoridae there was a basal split between Alligator and a branch that contained Paleosuchus and Caiman. The analyses also provided molecular estimates of various divergences applying recently established crocodylian and outgroup fossil calibration points. Molecular estimates based on amino acid data placed the divergence between Crocodylidae and Alligatoridae at 97-103 million years ago and that between Alligator and Caiman/Paleosuchus at 65-72 million years ago. Other crocodilian divergences were placed after the Cretaceous-Tertiary boundary. Thus, according to the molecular estimates, three extant crocodylian lineages have their roots in the Cretaceous. Considering the crocodylian diversification in the Cretaceous the molecular datings suggest that the extinction of the dinosaurs was also to some extent paralleled in the crocodylian evolution. However, for whatever reason, some crocodylian lineages survived into the Tertiary.