Understanding the distribution of genetic diversity among individuals, populations and gene pools is crucial for the efficient management of germplasm collections and breeding programs. Diversity analysis is routinely carried out using sequencing of selected gene(s) or molecular marker technologies. Here we report on the development of Diversity Arrays Technology (DArT) for pigeonpea (Cajanus cajan) and its wild relatives. DArT tests thousands of genomic loci for polymorphism and provides the binary scores for hundreds of markers in a single hybridization-based assay. We tested eight complexity reduction methods using various combinations of restriction enzymes and selected PstI/HaeIII genomic representation with the largest frequency of polymorphic clones (19.8%) to produce genotyping arrays. The performance of the PstI/HaeIII array was evaluated by typing 96 accessions representing nearly 20 species of Cajanus. A total of nearly 700 markers were identified with the average call rate of 96.0% and the scoring reproducibility of 99.7%. DArT markers revealed genetic relationships among the accessions consistent with the available information and systematic classification. Most of the diversity was among the wild relatives of pigeonpea or between the wild species and the cultivated C. cajan. Only 64 markers were polymorphic among the cultivated accessions. Such narrow genetic base is likely to represent a serious impediment to breeding progress in pigeonpea. Our study shows that DArT can be effectively applied in molecular systematics and biodiversity studies.

;The organisation of plant microtubules into distinct arrays during the cell cycle requires interactions with partner proteins. Having recently identified a 90-kDa phospholipase D (PLD) that associates with microtubules and the plasma membrane [Gardiner et al. (2001) Plant Cell 13: 2143], we exposed seeds and young seedlings of Arabidopsis to 1-butanol, a specific inhibitor of PLD-dependent production of the signalling molecule phosphatidic acid (PA). When added to agar growth media, 0.2% 1-butanol strongly inhibited the emergence of the radicle and cotyledons, while 0.4% 1-butanol effectively blocked germination. When normal seedlings were transferred onto media containing 0.2% and 0.4% 1-butanol, the inhibitor retarded root growth by about 40% and 90%, respectively, by reducing cell elongation. Inhibited plants showed significant swelling in the root elongation zone, bulbous or branched root hairs, and modified cotyledon morphology. Confocal immunofluorescence microscopy of root tips revealed that 1-butanol disrupted the organisation of interphase cortical microtubules. Butanol isomers that do not inhibit PLD-dependent PA production, 2-and 3-butanol, had no effect on seed germination, seedling growth, or microtubule organisation. We propose that production of PA by PLD may be required for normal microtubule organisation and hence normal growth in Arabidopsis.

The organization of microtubule arrays in the plant cell cortex involves interactions with the plasma membrane, presumably through protein bridges. We have used immunochemistry and monoclonal antibody 6G5 against a candidate bridge protein, a 90-kD tubulin binding protein (p90) from tobacco BY-2 membranes, to characterize the protein and isolate the corresponding gene. Screening an Arabidopsis cDNA expression library with the antibody 6G5 produced a partial clone encoding phospholipase D (PLD), and a full-length gene was obtained by sequencing a corresponding expressed sequence tag clone. The predicted protein of 857 amino acids contains the active sites of a phospholipidmetabolizing enzyme and a Ca 2 ؉-dependent lipid binding domain and is identical to Arabidopsis PLD ␦. Two amino acid sequences obtained by Edman degradation of the tobacco p90 are identical to corresponding segments of a PLD sequence from tobacco. Moreover, immunoprecipitation using the antibody 6G5 and tobacco BY-2 protein extracts gave significant PLD activity, and PLD activity of tobacco BY-2 membrane proteins was enriched 6.7-fold by tubulin-affinity chromatography. In a cosedimentation assay, p90 bound and decorated microtubules. In immunofluorescence microscopy of intact tobacco BY-2 cells or lysed protoplasts, p90 colocalized with cortical microtubules, and taxol-induced microtubule bundling was accompanied by corresponding reorganization of p90. Labeling of p90 remained along the plasma membrane when microtubules were depolymerized, although detergent extraction abolished the labeling. Therefore, p90 is a specialized PLD that associates with membranes and microtubules, possibly conveying hormonal and environmental signals to the microtubule cytoskeleton.

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