Recent Progress in Development of Tnt1 Functional Genomics Platform for Medicago truncatula and Lotus japonicus in Bulgaria

Legumes, as protein-rich crops, are widely used for human food, animal feed and vegetable oil production. Over the past decade, two legume species, Medicago truncatula and Lotus japonicus, have been adopted as model legumes for genomics and physiological studies. The tobacco transposable element, Tnt1, is a powerful tool for insertional mutagenesis and gene inactivation in plants. A large collection of Tnt1-tagged lines of M. truncatula cv. Jemalong was generated during the course of the project 'GLIP': Grain Legumes Integrated Project, funded by the European Union (www.eugrainlegumes.org). In the project 'IFCOSMO': Integrated Functional and COmparative genomics Studies on the MOdel Legumes Medicago truncatula and Lotus japonicus, supported by a grant from the Ministry of Education, Youth and Science, Bulgaria, these lines are used for development of functional genomics platform of legumes in Bulgaria. This review presents recent advances in the evaluation of the M. truncatula Tnt1 mutant collection and outlines the steps that are taken in using the Tnt1-tagging for generation of a mutant collection of the second model legume L. japonicus. Both collections will provide a number of legume-specific mutants and serve as a resource for functional and comparative genomics research on legumes. Genomics technologies are expected to advance genetics and breeding of important legume crops (pea, faba bean, alfalfa and clover) in Bulgaria and worldwide.     

Growth and nitrogen fixation in Lotus japonicus and Medicago truncatula under NaCl stress: nodule carbon metabolism

Lotus japonicus and Medicago truncatula model legumes, which form determined and indeterminate nodules, respectively, provide a convenient system to study plant-Rhizobium interaction and to establish differences between the two types of nodules under salt stress conditions. We examined the effects of 25 and 50mM NaCl doses on growth and nitrogen fixation parameters, as well as carbohydrate content and carbon metabolism of M. truncatula and L. japonicus nodules. The leghemoglobin (Lb) content and nitrogen fixation rate (NFR) were approximately 10.0 and 2.0 times higher, respectively, in nodules of L. japonicus when compared with M. truncatula. Plant growth parameters and nitrogenase activity decreased with NaCl treatments in both legumes. Sucrose was the predominant sugar quantified in nodules of both legumes, showing a decrease in concentration in response to salt stress. The content of trehalose was low (less than 2.5% of total soluble sugars (TSS)) to act as an osmolyte in nodules, despite its concentration being increased under saline conditions. Nodule enzyme activities of trehalose-6-phosphate synthase (TPS) and trehalase (TRE) decreased with salinity. L. japonicus nodule carbon metabolism proved to be less sensitive to salinity than in M. truncatula, as enzymatic activities responsible for the carbon supply to the bacteroids to fuel nitrogen fixation, such as sucrose synthase (SS), alkaline invertase (AI), malate dehydrogenase (MDH) and phosphoenolpyruvate carboxylase (PEPC), were less affected by salt than the corresponding activities in barrel medics. However, nitrogenase activity was only inhibited by salinity in L. japonicus nodules.     

A Set of Lotus japonicus Gifu x Lotus burttii Recombinant Inbred Lines Facilitates Map-based Cloning and QTL Mapping

Model legumes such as Lotus japonicus have contributed significantly to the understanding of symbiotic nitrogen fixation. This insight is mainly a result of forward genetic screens followed by map-based cloning to identify causal alleles. The L. japonicus ecotype 'Gifu' was used as a common parent for inter-accession crosses to produce F2 mapping populations either with other L. japonicus ecotypes, MG-20 and Funakura, or with the related species L. filicaulis. These populations have all been used for genetic studies but segregation distortion, suppression of recombination, low polymorphism levels, and poor viability have also been observed. More recently, the diploid species L. burttii has been identified as a fertile crossing partner of L. japonicus. To assess its qualities in genetic linkage analysis and to enable quantitative trait locus (QTL) mapping for a wider range of traits in Lotus species, we have generated and genotyped a set of 163 Gifu × L. burttii recombinant inbred lines (RILs). By direct comparisons of RIL and F2 population data, we show that L. burttii is a valid alternative to MG-20 as a Gifu mapping partner. In addition, we demonstrate the utility of the Gifu × L. burttii RILs in QTL mapping by identifying an Nfr1-linked QTL for Sinorhizobium fredii nodulation.     

Multiple origins of the determinate growth habit in domesticated common bean (Phaseolus vulgaris)

Background and Aims

The actual number of domestications of a crop is one of the key questions in domestication studies. Answers to this question have generally been based on relationships between wild progenitors and domesticated descendants determined with anonymous molecular markers. In this study, this question was investigated by determining the number of instances a domestication phenotype had been selected in a crop species. One of the traits that appeared during domestication of common bean (Phaseolus vulgaris) is determinacy, in which stems end with a terminal inflorescence. It has been shown earlier that a homologue of the arabidopsis TFL1 gene – PvTFL1y – controls determinacy in a naturally occurring variation of common bean.

Methods

Sequence variation was analysed for PvTFL1y in a sample of 46 wild and domesticated accessions that included determinate and indeterminate accessions.

Key Results

Indeterminate types – wild and domesticated – showed only synonymous nucleotide substitutions. Determinate types – observed only among domesticated accessions – showed, in addition to synonymous substitutions, non-synonymous substitutions, indels, a putative intron-splicing failure, a retrotransposon insertion and a deletion of the entire locus. The retrotransposon insertion was observed in 70 % of determinate cultivars, in the Americas and elsewhere. Other determinate mutants had a more restricted distribution in the Americas only, either in the Andean or in the Mesoamerican gene pool of common bean.

Conclusions

Although each of the determinacy haplotypes probably does not represent distinct domestication events, they are consistent with the multiple (seven) domestication pattern in the genus Phaseolus. The predominance of determinacy in the Andean gene pool may reflect domestication of common bean prior to maize introduction in the Andes.     

Two Distinct Plastid Genome Configurations and Unprecedented Intraspecies Length Variation in the accD Coding Region in Medicago truncatula

We fully sequenced four and partially sequenced six additional plastid genomes of the model legume Medicago truncatula. Three accessions, Jemalong 2HA, Borung and Paraggio, belong to ssp. truncatula, and R108 to ssp. tricycla. We report here that the R108 ptDNA has a ∼45-kb inversion compared with the ptDNA in ssp. truncatula, mediated by a short, imperfect repeat. DNA gel blot analyses of seven additional ssp. tricycla accessions detected only one of the two alternative genome arrangements, represented by three and four accessions each. Furthermore, we found a variable number of repeats in the essential accD and ycf1 coding regions. The repeats within accD are recombinationally active, yielding variable-length insertions and deletions in the central part of the coding region. The length of ACCD was distinct in each of the 10 sequenced ecotypes, ranging between 650 and 796 amino acids. The repeats in the ycf1 coding region are also recombinationally active, yielding short indels in 10 regions of the reading frames. Thus, the plastid genome variability we report here could be linked to repeat-mediated genome rearrangements. However, the rate of recombination was sufficiently low, so that no heterogeneity of ptDNA could be observed in populations maintained by single-seed descent.     

Mineral nutrient imbalance, total antioxidants level and DNA damage in common bean (Phaseolus vulgaris L.) exposed to heavy metals

The present study aimed to analyze the biological effects induced by bioaccumulation of metals in common bean (Phaseolus vulgaris L.). Effects of mineral nutrient imbalance, total antioxidants level and DNA damage induced by accumulation of heavy metals, were investigated in bean seedlings treated with two selected metal concentrations for 7 days. Metal content is analyzed by inductively coupled plasma – atomic emission spectrometer (ICP-AES), for total antioxidants level assessment the Ferric-Reducing Antioxidant Power (FRAP) assay is used and Random Amplified Polymorphic DNA (RAPD) method was applied for investigation of DNA damages. The increasing metal concentration in the treatment medium changed synchronously metal content in samples, and decreased total antioxidant activity in all samples with exception only for samples treated with Ni and Cd. The obtained “DNA fingerprints” demonstrated that the increasing metal concentrations induced changes in RAPD profiles (disappearance and/or appearance of bands in comparison with untreated control samples). The highest number of missing bands was observed in samples treated with zinc (total 4 bands) and nickel (total 4 bands) at both concentrations. These results suggested that mineral nutrient imbalance is involved in changes of antioxidant levels and DNA damages of the seedlings, which may help to understand the mechanism of metal toxicity in plants.     

Evolution and microsynteny of the apyrase gene family in three legume genomes

Apyrases have been suggested to play important roles in plant nutrition, photomorphogenesis, and nodulation. To help trace the evolution of these genes in the legumes—and possibly, the acquisition of new functions for nodulation—apyrase-containing BACs were sequenced from three legume genomes. Genomic sequences from Medicago truncatula, Glycine max and Lotus japonicus were compared to one another and to corresponding regions in Arabidopsis thaliana. A phylogenetic analysis of apyrase homologs from these regions and sequences from other legume species, as well as other plant families, identified a potentially legume-specific clade that contains a well-characterized soybean ( G. soja) apyrase, Gs52, as well as homologs from Dolichos, Lotus , Medicago and Pisum. Sister clades contain homologs from members of Brassicaceae, Solanaceae, Poaceae and Fabaceae. Comparisons of rates of change at synonymous and nonsynonymous sites in the Gs52 and sister clades show rapid evolution in the potentially legume-specific Gs52 clade. The genomic organization of the apyrase-containing BACs shows evidence of gene duplication, genomic rearrangement, and gene conversion among Gs52 homologs. Taken together, these results suggest a scenario of local apyrase gene duplication in an ancestor of the legumes, followed by functional diversification and increased rates of change in the new genes, and further duplications in the Galegae (which include the genera Medicago and Pisum). The study also provides a detailed comparison of genomic regions between two model genomes which are now being sequenced ( M. truncatula and L. japonicus), and a genome from an economically important legume species ( G. max).Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by A. Kondorosi     

Effects of Temperature on Resistance in Phaseolus vulgaris Genotypes and on Development of Meloidogyne Species

Phaseolus vulgaris lines with heat-stable resistance to Meloidogyne spp. may be needed to manage root-knot nematodes in tropical regions. Resistance expression before and during the process of nematode penetration and development in resistant genotypes were studied at pre- and postinoculation temperatures of 24 °C and 24 °C, 24 °C and 28 °C, 28 °C and 24 °C, and 28 °C and 28 °C. Resistance was effective at all temperature regimes examined, with fewer nematodes in roots of a resistant line compared with a susceptible line. Preinoculation temperature did not modify resistance expression to later infections by root-knot nematodes. However, postinoculation temperatures affected development of Meloidogyne spp. in both the resistant and susceptible bean lines tested. The more rapid development of nematodes to adults at the higher postinoculation temperature of 28 °C in both bean lines suggests direct temperature effects on nematode development instead of on resistance expression of either of two gene systems. Also, resistance was stable at 30 °C and 32 °C.     

Combined inoculation with Glomus intraradices and Rhizobium tropici CIAT899 increases phosphorus use efficiency for symbiotic nitrogen fixation in common bean (Phaseolus vulgaris L.)

This study compared the response of common bean (Phaseolus vulgaris L.) to arbuscular mycorrhizal fungi (AMF) and rhizobia strain inoculation. Two common bean genotypes i.e. CocoT and Flamingo varying in their effectiveness for nitrogen fixation were inoculated with Glomus intraradices and Rhizobium tropici CIAT899, and grown for 50 days in soil–sand substrate in glasshouse conditions. Inoculation of common bean plants with the AM fungi resulted in a significant increase in nodulation compared to plants without inoculation. The combined inoculation of AM fungi and rhizobia significantly increased various plant growth parameters compared to simple inoculated plants. In addition, the combined inoculation of AM fungi and rhizobia resulted in significantly higher nitrogen and phosphorus accumulation in the shoots of common bean plants and improved phosphorus use efficiency compared with their controls, which were not dually inoculated. It is concluded that inoculation with rhizobia and arbuscular mycorrhizal fungi could improve the efficiency in phosphorus use for symbiotic nitrogen fixation especially under phosphorus deficiency.     

Evolutionary history of the Tip100 transposon in the genus Ipomoea

Tip100 is an Ac-like transposable element that belongs to the hAT superfamily. First discovered in Ipomoea purpurea (common morning glory), it was classified as an autonomous element capable of movement within the genome. As Tip100 data were already available in databases, the sequences of related elements in ten additional species of Ipomoea and five commercial varieties were isolated and analyzed. Evolutionary analysis based on sequence diversity in nuclear ribosomal Internal Transcribed Spacers (ITS), was also applied to compare the evolution of these elements with that of Tip100 in the Ipomoea genus. Tip100 sequences were found in I. purpurea, I. nil, I. indica and I. alba, all of which showed high levels of similarity. The results of phylogenetic analysis of transposon sequences were congruent with the phylogenetic topology obtained for ITS sequences, thereby demonstrating that Tip100 is restricted to a particular group of species within Ipomoea. We hypothesize that Tip100 was probably acquired from a common ancestor and has been transmitted vertically within this genus.     

Amplification, contraction and genomic spread of a satellite DNA family (E180) in Medicago (Fabaceae) and allied genera

Background and Aims

Satellite DNA is a genomic component present in virtually all eukaryotic organisms. The turnover of highly repetitive satellite DNA is an important element in genome organization and evolution in plants. Here we assess the presence and physical distribution of the repetitive DNA E180 family in Medicago and allied genera. Our goals were to gain insight into the karyotype evolution of Medicago using satellite DNA markers, and to evaluate the taxonomic and phylogenetic signal of a satellite DNA family in a genus hypothesized to have a complex evolutionary history.

Methods

Seventy accessions from Medicago, Trigonella, Melilotus and Trifolium were analysed by PCR to assess the presence of the repetitive E180 family, and fluorescence in situ hybridization (FISH) was used for physical mapping in somatic chromosomes.

Key Results

The E180 repeat unit was PCR-amplified in 37 of 40 taxa in Medicago, eight of 12 species of Trigonella, six of seven species of Melilotus and in two of 11 Trifolium species. Examination of the mitotic chromosomes revealed that only 13 Medicago and two Trigonella species showed FISH signals using the E180 probe. Stronger hybridization signals were observed in subtelomeric and interstitial loci than in the pericentromeric loci, suggesting this satellite family has a preferential genomic location. Not all 13 Medicago species that showed FISH localization of the E180 repeat were phylogenetically related. However, nine of these species belong to the phylogenetically derived clade including the M. sativa and M. arborea complexes.

Conclusions

The use of the E180 family as a phylogenetic marker in Medicago should be viewed with caution. Its amplification appears to have been produced through recurrent and independent evolutionary episodes in both annual and perennial Medicago species as well as in basal and derived clades.     

Evolutionary history of the Afro-Madagascan Ixora species (Rubiaceae): species diversification and distribution of key morphological traits inferred from dated molecular phylogenetic trees

Background and Aims

Previous work on the pantropical genus Ixora has revealed an Afro-Madagascan clade, but as yet no study has focused in detail on the evolutionary history and morphological trends in this group. Here the evolutionary history of Afro-Madagascan Ixora spp. (a clade of approx. 80 taxa) is investigated and the phylogenetic trees compared with several key morphological traits in taxa occurring in Madagascar.

Methods

Phylogenetic relationships of Afro-Madagascan Ixora are assessed using sequence data from four plastid regions (petD, rps16, rpoB-trnC and trnL-trnF) and nuclear ribosomal external transcribed spacer (ETS) and internal transcribed spacer (ITS) regions. The phylogenetic distribution of key morphological characters is assessed. Bayesian inference (implemented in BEAST) is used to estimate the temporal origin of Ixora based on fossil evidence.

Key Results

Two separate lineages of Madagascan taxa are recovered, one of which is nested in a group of East African taxa. Divergence in Ixora is estimated to have commenced during the mid Miocene, with extensive cladogenesis occurring in the Afro-Madagascan clade during the Pliocene onwards.

Conclusions

Both lineages of Madagascan Ixora exhibit morphological innovations that are rare throughout the rest of the genus, including a trend towards pauciflorous inflorescences and a trend towards extreme corolla tube length, suggesting that the same ecological and selective pressures are acting upon taxa from both Madagascan lineages. Novel ecological opportunities resulting from climate-induced habitat fragmentation and corolla tube length diversification are likely to have facilitated species radiation on Madagascar.