Identification of softness syndrome-associated candidate genes and DNA sequence variation in the sea squirt, Halocynthia roretzi.

The mortality of sea squirts, Halocynthia roretzi, with softness syndrome threatens the sea squirt aquaculture industry in Asian countries. The molecular approach to understanding the pathogenesis of softness syndrome began with differential gene expression analysis of tissues from normal and dying organisms. In the present study, we show that the expression of Halocynthia roretzi metalloproteinase (HrMMP) was significantly upregulated in the tissues of dying organisms through screening of differentially expressed genes, reverse transcription-polymerase chain reaction (RT-PCR), and real-time PCR. HrMMP is composed of 482 amino acids, contains a conserved domain found in the astacin family, and has typical metalloproteinase activity. To discriminate between the differential expression of the HrMMP gene in normal and dying organisms, we cloned the HrMMP gene promoter and identified a polymorphism in the HrMMP promoter region that resulted in distinct polymorphisms (G/T) at position - 308 bp. These results suggest that organisms with the GT genotype may have more resistance to softness syndrome than those with the TT genotype. These findings suggest that the HrMMP promoter polymorphism may be associated with an increased risk of softness syndrome in cultivated sea squirts and should be evaluated as a candidate molecular marker for the selective breeding of softness syndrome-resistant sea squirts.