Genetic, Biochemical, and Morphological Diversity of the Legume Biofuel Tree Pongamia pinnata

Pongamia pinnata is regarded as a sustainable biofuel feedstock of the future because of its abundant production of oilrich seeds, tolerance to abiotic stress, and ability to undergo biological nitrogen fixation (minimizing nitrogen inputs). However, it needs extensive domestication through selection and genetic improvement. Owing to its outcrossing nature, Pongamia displays large phenotypic diversity, which is advantageous for selection of desirable phenotypes but problematic for plantation management. In this study, variation was evaluated for seed mass, oil content, and oil composition. To evaluate genetic diversity and to lay the basis for a molecular breeding approach we developed second generation sequencing (2GS)-derived ISSR markers (Pongamia Inter-Simple Sequence Repeats; PISSR). The special feature of PISSRs is that the number of nucleotide repeats and the 5’ and 3’ nucleotide extensions were not arbitrarily chosen, but were based on Pongamia genomic sequences obtained from a NGS (Illumina®) database. Amplification products were resolved by polyacrylamide gel electrophoresis and silver staining or automated capillary electrophoresis to yield distinct and reproducible profiles. Polymorphic bands were excised from polyacrylamide gels and sequenced to reveal similarity to DNA sequences from other legumes. We demonstrated: 1) an abundance of nucleotide core repeats in the Pongamia genome, 2) large genetic and phenotypic diversity among randomly sampled Pongamia trees, 3) restricted diversity in progeny derived from a single mature tree; 4) stability of PISSR markers in Pongamia clones; and 5) genomic DNA sequences within PISSR markers. PISSRs provide a valuable biotechnology tool for assessment of genetic diversity, gene tagging and molecular breeding in Pongamia pinnata.