Chloroplast genome analysis of Angiosperms and phylogenetic relationships among Lamiaceae members with particular reference to teak (Tectona grandis L.f).

Availability of comprehensive phylogenetic tree for flowering plants which includes many of the economically important crops and trees is one of the essential requirements of plant biologists for diverse applications. It is the first study on the use of chloroplast genome of 3265 Angiosperm taxa to identify evolutionary relationships among the plant species. Sixty genes from chloroplast genome was concatenated and utilized to generate the phylogenetic tree. Overall the phylogeny was in correspondence with Angiosperm Phylogeny Group (APG) IV classification with very few taxa occupying incongruous position either due to ambiguous taxonomy or incorrect identification. Simple sequence repeats (SSRs) were identified from almost all the taxa indicating the possibility of their use in various genetic analyses. Large proportion (95.6%) of A/T mononucleotide was recorded while the di, tri, tetra, penta and hexanucleotide amounted to less than 5%. Ambiguity of the taxonomic status of Tectona grandis L.f was assessed by comparing the chloroplast genome with closely related Lamiaceae members through nucleotide diversity and contraction/expansion of inverted repeat regions. Although the gene content was highly conserved, structural changes in the genome was evident. Phylogenetic analysis suggested that Tectona could qualify for a subfamily Tectonoideae. Nucleotide diversity in intergenic and genic sequences revealed prominent hyper-variable regions such as, rps16-trnQ, atpH-atpI, psc4-psbJ, ndhF, rpl32 and ycf1 which have high potential in DNA barcoding applications.

Quantitative trait loci mapping for stomatal traits in interspecific hybrids of Eucalyptus.

Eucalyptus is an important industrial species with tolerance to drought and salt stress. Genetic improvement activities including quantitative trait loci (QTL) mapping for pulping and adventitious rooting traits are in progress, but no information is available on the genomic regions on adaptive traits such as stomatal characteristics. In this study, an interspecific cross between Eucalyptus tereticornis and E. grandis was generated for the development of genetic map and QTL identification for stomatal traits. Simple sequence repeats (SSRs), inter-simple sequence repeats (ISSRs) and sequence related amplified polymorphism (SRAP)markers were used for genotyping the F1 individuals. Parent-specific geneticmaps (female, 1023.56 cM;male, 1049.64cM) and consensus map (1049.4 cM) were developed. QTL analysis was carried out to identify the chromosomal regions affecting stomatal density, area and pore length in adaxial and abaxial leaf surface. Seven QTLs were identified with phenotypic variation of 11.36 to 27.30% for stomatal density, area and pore length. Correlation of stomatal traits when combined with growth and wood properties would have greater implications for generation of stress tolerant eucalypt hybrids with higher productivity and adaptability.

Hybrid purity assessment in Eucalyptus F1 hybrids using microsatellite markers.

The worldwide expansion of hybrid breeding and clonal forestry is to meet the demands of paper pulp and bioenergy. Although India was one of the pioneers in hybrid production of eucalypts only recently the hybrid clonal forestry is gaining momentum. Inter-specific hybrids are being produced to exploit the hybrid vigor of F1 individuals. Quality control genotyping for hybrid purity and parentage confirmation at the early stage is one of the essential criteria for clonal propagation and field trails for the assessment of growth performance. Eucalyptus being a obligatory outcrossed species with potential to self pollination, possibilities of pollen contamination are high. Hence, in the present study, Eucalyptus camaldulensis × E. tereticornis inter-specific hybrids were genotyped using 25 fluorescent labeled microsatellite markers available in public domain. Multiplex loading of PCR products was performed successfully for most of the microsatellite loci. Hybrid purity index was calculated and parentage was confirmed. Hybrid purity values ranged from 85 to 100 % showed the efficiency of controlled pollination techniques. A subset of six fully informative simple sequence repeats was identified for routine quality control genotyping for these hybrids. Detection of non-essential genotypes observed among the hybrid seedlings proved the significance of hybrid purity tests and the false hybrids were removed at the seedling stage. The hybrids with proven hybridity will be used for generation of genetic linkage, discovery of quantitative trait loci and the individuals with high productivity can enter into mass clonal multiplication.

Impact of sugar factory effluent on the growth and biochemical characteristics of terrestrial and aquatic plants.

The physico-chemical characteristics of sugar industry effluent were measured and some were found to be above those limits permissible in the Indian irrigation water standard. A pot study was initially conducted to study the effects of different concentrations (20%, 40%, 60%, 80% and 100%) of sugar factory effluent on seed germination, seedling growth and biochemical characteristics of green gram and maize. A similar study was also carried out using the aquatic plants, water hyacinth and water lettuce. The higher effluent concentrations (above 60%) were found to affect plant growth, but diluted effluent (up to 60%) favored seedling growth.

Eucalyptus microsatellites mined in silico: survey and evaluation.

Eucalyptus is an important short rotation pulpy woody plant, grown widely in the tropics. Recently, many genomic programmes are underway leading to the accumulation of voluminous genomic and expressed sequence tag sequences in public databases. These sequences can be utilized for analysis of simple sequence repeats (SSRs) and single nucleotide polymorphism (SNPs) available in the transcribed genes. In this study, in silico analysis of 15,285 sequences representing partial and full-length mRNA from Eucalyptus species for their use in developing SSRs or microsatellites were carried out. A total of 875 EST-SSRs were identified from 772 SSR containing ESTs. Motif size of 6 for dinucleotide and 5 for trinucleotide, tetranucleotide, and pentanucleotides were considered in locating the microsatellites. The average frequency of identified SSRs was 12.9%. The dinucleotide repeats were the most abundant among the dinucleotide, trinucleotide and tetranucleotide motifs and accounted for 50.9% of the Eucalyptus genome. Primer designing analysis showed that 571 sequences with SSRs had sufficient flanking regions for polymerase chain reaction (PCR) primer synthesis. Evaluation of the usefulness of the SSRs showed that EST-derived SSRs can generate polymorphic markers as all the primers showed allelic diversity among the 16 provenances of E. tereticornis.

Determination of inter- and intra-species genetic relationships among six Eucalyptus species based on inter-simple sequence repeats (ISSR).

Eucalyptus is the most economically important hardwood plantation tree cultivated in tropical and subtropical countries. Inter-simple sequence repeat (ISSR) markers were used to evaluate genetic relationships within and between individuals of six Eucalyptus species. A total of 583 loci (265 to 1535 bp) were amplified from 149 individuals belonging to the six Eucalyptus species using seven ISSR primers (two to three nucleotide repeats anchored with one or two nucleotides at the 3' or 5' region). The ISSR fragments indicated significant polymorphism and genetic diversity among the individuals. Cluster analysis and principal component analysis revealed the occurrence of wide genetic diversity among populations of E. tereticornis Sm., E. camaldulensis Dehnh. and E. urophylla S.T. Blake and narrow genetic diversity among populations of E. citriodora Hook. and E. grandis W. Hill ex Maiden. Genetic diversity was high in E. tereticornis Sm. (47.27%) and low in E. citriodora (18.64%). Maximum Nei's genetic identity (0.897) was observed between E. camaldulensis and E. tereticornis species, whereas maximum genetic diversity (0.286) was found between individuals of E. citriodora and E. grandis.

Genetic analyses of casuarinas using ISSR and FISSR markers.

Inter simple sequence repeat polymerase chain reaction (ISSR-PCR) was used for the genetic analysis of the six species of Allocasuarina, five species of Casuarina and 12 superior performing selections of C. equisetifolia L. We also fingerprinted C. equisetifolia L. selections using Fluorescent-ISSR-PCR (FISSR-PCR), an improvised ISSR-PCR assay. The ISSR analysis provided information on the frequency of various simple sequence repeats in the casuarina genome. The di-nucleotide repeats were more common, among which (CA)n and its complementary nucleotide (GT),, repeat motifs amplified relatively higher number of bands with an average of 6.0+/-3.5 and 6.3+/-1.8 respectively. Eleven species of casuarinas were amplified with 10 primers anchored either at 5' or 3' end. A total of 253 PCR products were obtained and all were polymorphic, out of which 48 were specific to Allocasuarina and 36 were specific to Casuarina genus. Genetic similarity among the species was 0.251. A UPGMA dendrogram grouped all the Casuarina species together. The 12 superior performing selections of C. equisetifolia L. produced 57 polymorphic ISSR markers while the FISSR assay revealed 105 polymorphic markers. The primer CRR(ATT)4 distinguished all the selections. DNA profiles obtained with ISSR and FISSR assays would serve as a reference library for the establishment of clonal identity in casuarinas.