Qunyi Jiang

Classical and Molecular Genetics of the Model Legume Lotus japonicus

The model legume Lotus japonicus was demonstrated to be amenable to classical and molecular genetic analysis, providing the basis for the genetic dissection of the plant processes underlying nodulation and nitrogen fixation. We have developed an efficient method for the sexual hybridization of L. japonicus and obtained F1 progeny derived from a cross of L. japonicus B-129-S9 Gifu x B-581 Funakura. Over half of the cross-pollinations resulted in fertile hybrid seed, which were confirmed morphologically and by single arbitrary primer DNA amplification polymorphisms using the DAF technique. Molecular and morphological markers segregated in true Mendelian fashion in a F2 population of 100 plants. Several DAF loci were linked using the MAPMAKER software to create the first molecular linkage groups of this model legume. The mapping population was advanced to generate a set of immortal recombinant inbred lines (F6; RILs), useful for sharing plant material fixed genetically at most genomic regions. Morphological loci for waved stem shape (Ssh), dark leaf color (Lco), and short flowering period (Fpe) were inherited as single dominant Mendelian loci. DAF markers were dominant and were detected between Gifu and Funakura at about one per primer, suggesting that the parents are closely related. One polymorphism (270G generated by single octomer primer 8.6m) was linked to a morphological locus controlling leaf coloration. The results demonstrate that (i) Lotus japonicus is amenable to diploid genetic analysis, (ii) morphological and molecular markers segregate in true diploid fashion, (iii) molecular polymorphisms can be obtained at a reasonable frequency between the related Gifu and Funakura lines, and iv) the possibility exists for map-based cloning, marker assisted selection and mapping of symbiotic mutations through a genetic and molecular map.

Genetic analysis of ethylene regulation of legume nodulation.

The gaseous hormone ethylene has multiple roles in plant development and responses to external cues. Among these is the regulation of ‘Rhizobium’-induced nodulation in legumes. Extensive descriptive literature exists, but has been expanded to allow more mechanistic analysis through the application of genetics. Both mutants and transgenics displaying ethylene insensitivity have now been described, suggesting an intimate interplay of ethylene response, plant development and nodulation.

The REDUCED LEAFLET Genes Encode Key Components of the trans-Acting Small Interfering RNA Pathway and Regulate Compound Leaf and Flower Development in Lotus japonicus

The endogenous trans-acting small interfering RNA (ta-siRNA) pathway plays a conserved role in adaxial-abaxial patterning of lateral organs in simple-leafed plant species. However, its function in compound-leafed species is largely unknown. Using the compound-leafed species Lotus japonicus, we identified and characterized two independent mutants, reduced leaflet1 (rel1) and rel3, whose most conspicuous defects in compound leaves are abaxialized leaflets and reduction in leaflet number. Concurrent mutations in REL genes also compromise flower development and result in radial symmetric floral organs. Positional cloning revealed that REL1 and REL3 encode the homologs of Arabidopsis (Arabidopsis thaliana) SUPPRESSOR OF GENE SILENCING3 and ARGONAUTE7/ZIPPY, respectively, which are key components of the ta-siRNA pathway. These observations, together with the expression and functional data, demonstrated that the ta-siRNA pathway plays conserved yet distinct roles in the control of compound leaf and flower development in L. japonicus. Moreover, the phenotypic alterations of lateral organs in ta-siRNA-deficient mutants and the regulation of downstream targets by the ta-siRNA pathway in L. japonicus were similar to those in the monocots but different from Arabidopsis, indicating many parallels between L. japonicus and the monocots in the control of lateral organ development by the ta-siRNA pathway.

Kinetics of Nodule Development in Glycine soja

Nodule development in the interaction of Glycine soja Sieb. & Zucc. PI468.397 with Bradyrhizobium japonicum USDA110 was studied by hypochlorite clearing and methylene blue staining. Even the earliest stages of nodule development could be observed. The entire length of the primary root was examined up to 15 d postinoculation. Markedly curled root hairs and the first cell divisions in the hypodermal layer (stage I) were observed 2 d postinoculation, and by 3 d cell division activity had spread to the outer layers of the cortex (stage II). Cortical cell division centers not associated with curled root hairs, frequently observed in soybean (Glycine max [L.] Merr.), were very rare in G. soja. The cortical cell division centers that had developed a well-defined nodule meristem (at or beyond stage IV) by 6 d postinoculation continued to develop, but the less-advanced stages became arrested. Almost all nodules developed near the position of the root tip at the time of inoculation. In the parts of the root that developed after inoculation, regions with a high density of markedly curled root hairs per root length were observed. The percentage of the curled root hairs associated with cortical cell division centers, however, declined with each successive peak. Regulation of nodule development in G. soja was similar to that previously reported in soybean, although the rate of nodule development was slower.

Genetic and genomic analysis of the tree legume Pongamia pinnata as a feedstock for biofuels.

The tree legume Pongamia {Pongamia pinnata (L.) Pierre [syn. Millettia pinnata (L.) Panigrahi]} is emerging as an important biofuels feedstock. It produces about 30 kg per tree per year of seeds, containing up to 55% oil (w/v), of which approximately 50% is oleic acid (C18:1). The capacity for biological N fixation places Pongamia in a more sustainable position than current nonlegume biofuel feedstocks. Also due to its drought and salinity tolerance, Pongamia can grow on marginal land not destined for production of food. As part of the effort to domesticate Pongamia our research group at The University of Queensland has started to develop specific genetic and genomic tools. Much of the preliminary work to date has focused on characterizing the genetic diversity of wild populations. This diversity is reflective of the outcrossing reproductive biology of Pongamia and necessitates the requirement to develop clonal propagation protocols. Both the chloroplast and mitochondrial genomes of Pongamia have been sequenced and annotated (152,968 and 425,718 bp, respectively), with similarities to previously characterized legume organelle genomes. Many nuclear genes associated with oil biosynthesis and nodulation in Pongamia have been characterized. The continued application of genetic and genomic tools will support the deployment of Pongamia as a sustainable biofuel feedstock.

Nodulation Control in Legumes

Nodulation and concomitant symbiotic nitrogen fixation are critical for the productivity of the legume, yielding food, feed and fuel. The nodule number in legumes is regulated by numerous factors including the number and efficiency of the interacting Rhizobium bacteria and abiotic stresses as well as endogenous processes involving phytohormones, nodulation reception systems and autoregulation of nodulation (AON; Kinkema et al., 2006). The original discovery of the AON-controlling LRR receptor kinases, GmNARK/ LjHAR1/MtSUNN, which is active in leaf tissue of several legu-mes, now has led to an analysis of the mechanism underlying the signal transduction.

Functional Genomics of the Regulation of Nodule Number in Legumes

Peter M. Gresshoff, Gustavo Gualtieri, Titeki Laniya, Arief Indrasumunar, Akira Miyahara, Sureeporn Nontachaiyapoom, Tim Wells, Bandana Biswas, Pick Kuen Chan, Paul Scott, M. Kinkema, M. Djordjevic, Dana Hoffmann, Lisette Pregelj, Diana M. Buzas, Dong Xi Li, Artem Men, Qunyi Jiang, Cheol-Ho Hwang and Bernard J. Carroll ARC Centre of Excellence for Integrative Legume Research; School of Life Sciences, and School of Molecular and Microbial Sciences and LAFS, The University of Queensland, St. Lucia, Brisbane QLD 4072, AGRF; Genome Interaction Group, RSBS, ANU, Canberra, ACT, Australia.