Molecular Cloning of Dormancy-associated MADS-box Gene Homologs and Their Characterization during Seasonal Endodormancy Transitional Phases of Japanese Pear

Abstract

To understand the role of the MIKC-type dormancy-associated MADS-box (DAM) genes in the regulation of endodormancy in japanese pear (Pyrus pyrifolia), we isolated two DAM genes from ‘Kosui’ and characterized their expression throughout the seasonal endodormancy phases in ‘Kosui’, as well as in TP-85–119 taiwanese pear (P. pyrifolia), which is a less dormant type. Several copies of the corresponding DAM genes are present in the P. pyrifolia genome. Rapid amplification of cDNA ends enabled the isolation of two full-length cDNAs, designated as PpMADS13–1 and PpMADS13–2, with complete open reading frames encoding 227 and 234 amino acids, respectively. Multialignment of the two ‘Kosui’ and the database DAM genes (based on the deduced amino acid sequences) showed that PpMADS13–1 and PpMADS13–2 were highly identical to the Rosaceae DAM genes and encoded the conserved domains characteristic of other MIKC-type MADS-box genes. The phylogenetic relationships showed that PpMADS13–1 and PpMADS13–2 were more closely related to the Prunus DAM, though they formed a unique subclade. The specific expression analysis of PpMADS13–1 and PpMADS13–2 by real-time polymerase chain reaction showed that both DAM genes are gradually down-regulated concomitant with endodormancy breaking. PpMADS13–1 and PpMADS13–2 showed similar fluctuations in expression patterns, although PpMADS13–2 was more highly expressed relative to PpMADS13–1. The expression of PpMADS13–1 and PpMADS13–2 in the less dormant taiwanese pear, TP-85–119, was quite low (nearly zero level), which is consistent with a down-regulated pattern of expression of the DAM genes in japanese pear, peach (Prunus persica), and japanese apricot (Prunus mume). Differential genomic DNA methylation patterns detected in PpMADS13–1 and PpMADS13–2 were not concomitant with seasonal endodormancy transition phases, suggesting that DNA methylation in these loci under investigation may not be linked to endodormancy progression in ‘Kosui’.