Construction And Characterization Of Seed Transcriptome And Screening Of Polymorphic EST-SSR Loci In Millettia Pinnata

Millettia pinnata is a biofuel tree with a promising prospect for utilization. It belongs to the genus Millettia of the subfamily Papilionoideae and is a typical semi-mangrove plant which mainly distributed along the coastal areas in the southern China. Protection and utilization of germplasm resources of M. pinnata, which serves as one of the important bases for its planting and production of biodiesel, depends on in-depth elucidation of its genetic background. In this study, we performed high-throughput transcriptome sequencing based on the Illumina platform, followed by gene functional classification for M. pinnata seed tissues. Moreover, the polymorphism was evaluated for a set of potential EST-SSR(Simple Sequence Repeat) loci identified from the transcriptome data. The main results are as follows:(1) In this study, we chose the samples from three stages(histodifferentiation, seed filling, and dehydration) during seed development for RNA extraction and c DNA library construction. 80, 212, 402 reads were obtained by the paird-end sequencing, and were subsequently assembled into 53, 586 Unigenes. The mean length and total length of Unigenes were 787 nt and 42, 186, 440 nt, respectively. By blast against different databases, a total number of 39, 602(73.9%) Unigenes showed significant similarity to the Nr accenssions, of which more than 90% were annotated with protein accessions from legumes. The numbers of Unigenes annotated based on the Nt, Swiss-Prot, COG databases were 40, 938, 24, 078 and 13, 147, respectively; By performing GO term and KEGG pathway analyses, we found that there were 23, 489, 24, 052 and 23, 811 Unigenes involved in molecular function, cellular components and biological processes, respectively. Meanwhile, 21, 905 Unigenes were involved in 128 metabolic pathways including plant hormone signal transduction, plant-pathogen interactions, Spliceosome, RNA transport.(2) A total of 8, 212 potential EST-SSR loci were identified based on the Mp Seed-Unigene sequences. Most of these loci were two bases or three base repeats. We randomly chose 121 loci and designed primers according to theirs flanking sequences. Using DNA extracted from four individuals of M. pinnata, we performed PCR amplification and sequencing. The sequencing results showed that a total of 39 EST-SSR loci located in 35 Unigenes showed polymorphism in four individuals, which encompass 11 polymorphic EST-SSR loci located in genes related to lipid metabolismand 27 polymorphic EST-SSR loci in genes unrelated to lipid metabolism.The transcriptome data obtained in this study provided a solid basis for candidate gene cloning, functional analysis and transgenic applications. The validated polymorphic EST-SSR loci could not only be used for genetic diversity analysis, but also used in correlation analysis between molecular markers and phenotypic traits to find out polymorphic EST-SSR loci related to certain oil properties in M. pinnata. This will facilitate more convenient evaluation of M. pinnata germplasms.