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Cloning and Analysis of a MADS‐box Gene Expressed in Poa annua MD‐2 Apices during Floral Meristem Formation

Poa annua MD‐2 is a day‐neutral, annual grass, which flowers 25–35 d after planting under controlled conditions. Although the physiology and anatomy of flowering is fairly well understood, the genetic regulation of flowering in P. annua has not been studied. MADS‐box genes regulate reproductive development in other plants and are candidate genes for regulating flowering in P. annua. The purpose of this study was to isolate MADS‐box genes expressed in P. annua MD‐2 floral meristems. Degenerate primers based on the MADS‐box sequences of maize and rice were used to amplify a fragment of ca. 170 bp from MD‐2 genomic DNA. Plasmids containing inserts were sequenced and three of them (named PaMADS) were found to be 89%–93% identical to MADS sequences from maize. A cDNA library was constructed from 5 mg of MD‐2 floral meristem tissue between the transition and the panicle/branch primordial initiation stages. PaMADS sequence was used to screen the cDNA library and one full‐length MADS‐box gene named PaMADS1 was isolated. PaMADS1 is 1159 bp in length and encodes a 258 amino acid protein which shares 54.4% identity with AGL6 from Arabidopsis thaliana. The MADS‐box motif of PaMADS1 is identical with those of TaMADS#12 from wheat, ZAG3 from maize, and OsMADS6 from rice, and 98% identical with AGL6 and 83.0% with AGL13 from A. thaliana. The K‐box of PaMADS1 has 88.1%–94.1% identity with those of ZAG3, ZAG5, TaMADS#12, and OsMADS6, but only 60.4% identity with AGL6 and 53.5% with AGL13. Northern blot analysis indicated that PaMADS1 is a floral‐specific gene and is highly expressed at panicle/branch‐primordia‐initiation and prepollen stages. Southern hybridization indicated that there are ca. 10 copies of MADS‐box genes in both P. annua and Poa pratensis.