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Cloning and Identification of Highly Expressed Genes in Barley Lemma and Palea

^a Department of Agronomy, University of Wisconsin, Madison, WI 53706
^b USDA-ARS, Cereal Crops Research Unit, 501 Walnut St., Madison, WI 53726

View larger version (166K): [in a new window]   Fig. 1. Lemma and palea organs covering developing kernels of Morex barley. Left, lemma completely covers abaxial surface of kernel. The awn is continuous with the lemma. Center, palea covers most of the adaxial surface and is partially covered by the lemma. Ovary epithelial hairs (OEH) protrude above the palea tip. Right, lemma and palea teased apart from the developing kernel exposing the pericarp.

View larger version (70K): [in a new window]   Fig. 2. Differential screening of a subtracted lemma/palea cDNA library from elongating stage. Duplicate filter arrays of random clones (probes) from the elongating stage lemma/palea library were hybridized with 32P-labeled cDNA populations of 106 cpm/mL activity made from (A) forward subtracted cDNA (lemma/palea, tester; flag leaf, driver) and (B) reverse subtracted cDNA (flag leaf, tester; lemma/palea, driver). Clones in panel A showed differential hybridization and therefore represent mRNA sequences for genes highly expressed in the lemma/palea. The position of clones on the filter arrays is depicted in the table below.

View larger version (30K): [in a new window]   Fig. 3. Northern analysis of randomly selected clones confirms high expression levels in the lemma/palea compared with the flag leaf. Lemma and palea from the elongation, gelatinous and dough kernel stages were included to evaluate temporal expression patterns. Each lane contained 10 µg total RNA. The RNA blot was hybridized with 32P-labeled probes 1-5, 1-25, 1-52, 2-6, 2-24 and 3-13. EtBr stained agarose-formaldehyde gel is included at the bottom of each figure to show equal loading. 1, 2, and 3 represent pooled lemma/palea RNA from elongation, gelatinous and early dough stages of kernel development, respectively; FL, flag leaf.

View larger version (32K): [in a new window]   Fig. 4. Expression of selected cloned genes in mature lemma/palea and various leaves of barley. Each lane contained 10 µg total RNA. The RNA blot was hybridized with 32P-labeled 1-5, 1-14, 1-25, 1-52, 1-56, 1-107, 2-6, and 2-24 probes. EtBr stained agarose-formaldehyde gel is included at the bottom of each figure to show equal loading. L/P, pooled lemma/palea RNA from the middle (gelatinous) stage of kernel development; FL, flag leaf; 6, 6th leaf; 5, 5th leaf; 4, 4th leaf.