De novo sequencing and characterization of seed transcriptome of the tree legume Millettia pinnata for gene discovery and SSR marker development

Abstract

Pongamia (Millettia pinnata) is a promising biofuel crop with multiple merits. Breeding of ideal Pongamia germplasm for industrial application demands substantial progress in molecular biology of this legume species, which has been largely hampered by the paucity of its genomic data. In this study, we constructed and characterized a comprehensive seed transcriptome by the high-throughput Illumina sequencing technology. We obtained over 83 million high-quality reads, which were processed and assembled into 53,586 unigenes with a mean length of 787 bp. Among these unigenes, 39,602 (73.90 %) and 24,078 (44.93 %) showed significant similarity to proteins in the NCBI non-redundant and the Swiss-Prot protein databases, respectively. Of the annotated unigenes, 30,619 (57.14 %) were classified into 56 Gene Ontology categories. Furthermore, 21,905 (40.88 %) unigenes were assigned to 128 pathways in the Kyoto Encyclopedia of Genes and Genomes pathway database. A set of 364 unigenes involved in five pathways closely related to oil biosynthesis and accumulation were screened out as candidates for future functional analyses. On the other hand, 5710 expressed sequence tag-simple sequence repeats (EST-SSRs) were identified in 4951 unigenes with a density of one SSR every 7.39-kb sequence. One hundred EST-SSRs were randomly selected to validate amplification and to assess polymorphism among 12 Pongamia individuals. Eighty-two primer pairs successfully amplified DNA fragments and 17 of them detected polymorphism, in which the polymorphism information content values ranged from 0.14 to 0.57. This transcriptome dataset will serve as a valuable basis for studies on functional genomics, molecular genetics and molecular breeding of Pongamia.