Abstract：Crowtoe (Lotus corniculatus) is a legume forage with broad application prospects, but the efficiency of promotion and utilization is seriously affected by drought. In order to solve this problem, after many years of research, the high drought tolerance crowtoe germplasm B08 was selected by this team, finally. In order to further study and explore the drought-resistance mechanism of germplasm B08, suppression subtractive hybridization (SSH) technology was used to construct the positive and negative SSH cDNA library of B08 under drought stress. 600 positive clones were randomly selected from the forward and reverse libraries for sequencing analysis. The results showed that: (1) 574 and 595 high quality sequences were obtained in the forward and reverse libraries, with an average length of 207.5 bp and 242.7 bp, respectively. Finally, 632 unigenes sequences were obtained by assembling high quality sequence data, of which 178 were forward library and 454 reverse library. (2) The 632 unigenes were compared with public databases by BLAST, and 147 genes were annotated successfully. Among them, 80 were known genes, 67 were unknown genes, and 485 new genes were found. (3) The results of NCBI Non-Redundant Protein Sequence Database (nr) annotation showed that there were high homology with Populus trichocarpa, Malus domestica, Spinacia oleracea, Hordeum vulgare, Brassica napus, Cajanus cajan, Cicer arietinum, Coffea canephora and so on. The forward and reverse libraries were classified using Gene Ontology (GO) program. The genes involved in cell, cell part, organcell, binding, metabolic process, and catalytic activity, cellular process accounted for a large proportion. (4) The obtained unigenes involved the synthesis of plant photosynthesis, energy metabolism, hormone synthesis, protein metabolism, nucleic acid metabolism, ion transport and signal transduction. (5) Substances closely related to drought resistance of plants had been found, such as serine/threonine-protein phosphatase, calmodulin-like protein (CML), indole-3-acetic acid-induced protein ARG7, mitogen-activated protein kinase (MAPK) et al. The results of this study provides information for further cloning and verifying the drought-resistant genes, digging new drought-related genes, understanding the molecular mechanism of drought-resistance, and breeding new varieties with high drought-resistance.
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