Organ identity genes have been extensively studied in two species, Arabidopsis thaliana and Antirrhinum majus (snapdragon), and fall into three separate classes, A, B, and C, each controlling organ identity in two adjacent whorls (Weigel & Meyerowitz, 1994). The B loss-of-function mutants, replace second-whorl petals with sepals and third-whorl stamens with carpels and appear to be phenotypically similar to the barley mo mutants. Two Arabidopsis and two snapdragon B organ identity genes have been cloned and designated APETALA3 (AP3) and PISTILATA (PI) in Arabidopsis and DEFICIENS (DEF) and GLOBOSA (GLO) in snapdragon. These, and other organ identity genes, encode members of a gene family characterized by an amino-terminal DNA-binding and dimerization domain designated MADS for the first four members of this family, MCM1 (yeast), AG (Arabidopsis), DEF (Snapdragon), and SRF (mammals) (Schwarz-Sommer et al., 1990).
Here we report the isolation of MADS-like genes from barley and attempts to correlate the genes with mutant phenotypes.
Isolation and characterization of mutants. The multiovary mutants, designated mo,,a and mo,,b, were selected from fast neutron treated M2 generation field plantings. Plants were first observed as male sterile, but closer examination revealed the complete absence of anthers and an abnormal number of stigmas and carpels. A normal barley floret consists of a pistil, three anthers and two lodicules enclosed by a lemma and palea (Fig. 1). The mo,,a mutant lacks all anthers and has multiple carpels (Fig. 2A). The carpel structures appear to be complex and abnormal. The lodicules (monocot equivalent of petals) appear to be fairly normal, although enlarged. The mo,,b mutant also lacks all anthers which are replaced by normal looking carpels (Fig. 2B). The lodicules appear as twisted leaf-like structures resembling sepals.
Cloning and characterization of MADS-like genes. Ten barley genomic DNA lambda clones were isolated using a rice agamous-like gene as a probe. An 800 bp Hind III fragment was subcloned from one of these, designated pJS18-2 and used as an RFLP probe. This fragment was sequenced and shown to contain strong homology to the MADS domain.
The isolated lambda clones were digested with several restriction enzymes, blotted and probed sequentially with pJS18-2, and the Arabidopsis organ identity genes AGAMOUS (AG), APETALA3 (AP3), and PISTILATA (PI). The lambda clone 16 hybridized strongly to AG and AP3 while PI showed strong hybridization to lambda clone 22. These clones were analyzed in more detail.
The lambda 16 clone was restriction mapped and individual fragments used to test for polymorphism. Only the Sall fragment pJS68 was polymorphic and could be used for mapping (see below). Fragment 24 was shown to hybridize with a mRNA probe.
Genetic analyses. Female fertility was demonstrated for both mo,,a and mo,,b.The F2 segregation ratios were distorted for mo,,a (3/53 mo plants), but monogenic for mo,,b (1 1/50 mo plants). Allelism tests and mapping are in progress.
A lambda18 HindIII fragment, JS18-2, with homology to a rice agamous-like gene and shown to contain MADS-like sequences was used to map two loci on the barley RFLP map (Fig. 3). These loci, designated JS18A and JS18B, are located on chromosome 1P and the telomeric region of chromosome 7P. The lambda16Sall fragment, JS68, was polymorphic and 5 loci were mapped to chromosomes 1, 3 and 6 and designated JS68A to E (Fig. 3). One of the loci maps close to the n locus and may be the same as mo5.
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Figure legends :
Fig. 1. A normal barley floret consists of two lodicules (bottom), a pistil made up of a complex carpel and a biforked stigma (center) and three anthers with filaments.
Fig. 2A. The mutant mo,,a has normal looking, although enlarged lodicules (bottom) and a variable number of pistil-like structures. Note the abnormal-looking pistil structures including elongated styles and the one anther-like structure.
Fig. 2B. The mutant mo,,b has four normal looking pistils (bottom center and top) and two green leaf-like structures representing the lodicules (bottom left and right).
Fig. 3. A skeletal Steptoe x Morex RFLP map showing the locations of the JS18A and B and JS68A-E loci (in bold). The positions of N and mo5 are approximated.