Conventional morphological and pigementation traits, as well as disease resistance, have been used to distinguish the uniqueness of new soybean cultivars for purposes of plant variety protection. With increasing numbers of cultivars and a finite number of conventional characters, it has become apparent that such traits will not suffice to establish uniqueness. The objective of this work was to provide an initial evaluation of microsatellite or simple-sequence-repeat (SSR) DNA markers to develop unique DNA profiles of soybean genotypes. Microsatellites are DNA sequences such as (AT) n /(TA) n and (ATT) n /(TAA) n that are composed of tandemly repeated 2-5-basepair DNA core sequences. The DNA sequences flanking microsatellites are generally conserved allowing the selection of polymerase chain reaction (PCR) primers that will amplify the intervening SSR. Variation in the number of tandem repeats, "n", results in PCR product length differences. The SSR alleles present at three (AT) n /(TA) n and four (ATT) n /(TAA) n loci were determined in each of 96 diverse soybean genotypes. Between 11 and 26 alleles were found at each of the seven loci. Only two genotypes had identical SSR allelic profiles and these had very similar pedigrees. The gene diversity for the seven markers averaged 0.87 for all 96 genotypes and 0.74 for a subset of 26 North American cultivars. These are much higher than soybean gene diversity values obtained using RFLP markers, and are similar to the average values obtained for human microsatellite markers. SSR markers provide an excellent complement to the conventional markers that are currently used to characterize soybean genotypes.
ABSTRACT'To whom reprint requests should be addressed.Commonwealth Scientific and Industrial Research Organization, Australia.General procedures for maintenance of stock cultures, initiation of starter cultures, and preparation of inocula have been described (3). R meliloti strain 102F71 and R. trifolii strain TA1 were grown on modified B5 medium supplemented with 0.5 ,ug of biotin/L (3). All cultures were harvested in mid-exponential growth phase for preparation of inocula (3.5 days for Rhizobium spp. 32H1 and 3G4b4 and 36 h for R. meliloti 102F71 and R. trifolii TAI).Growth of Plants. Cowpea seeds (Vigna sinensis [L.] Endl. cv. California Black Eye) were obtained from W. Atlee Burpee Co., Warminster, PA. Seeds were surface-disinfected and grown in plastic growth pouches (Scientific Products, Evanston, IL) as described earlier for soybean (3). Plants were inoculated by adding 0.5 ml of inoculum (A620 nm = 0.08) in drops onto the lower portion of each root.Seeds of alfalfa (Medicago sativa L. cvs. Moapa and Vernal) were obtained from Dewine and Hamma Seed Company, Yellow Springs, OH. Seeds were surface-sterilized as described (3) and soaked for 2 h in sterile H20. Ten to 12 swollen seeds were transferred directly to each growth pouch. Plants were inoculated 48 h after transfer by adding 100 ,ul of inoculum (A620nm = 0.08) onto each root.Changes in the position ofepidermal cells on cowpea and alfalfa roots, relative to the RT4 and SERH marks (Fig. 1) made on the pouches, were measured as described for soybean (3).Seeds ofpeanut (Arachis hypogaea L. cv. Spanish) were obtained from W. Atlee Burpee Co., surface-sterilized for 5 min, and germinated as described (3). Healthy seedlings were transferred to growth pouches on the fourth day after transfer to plates and inoculated with 0.5 ml of inoculum (A620 = 0.08) when they possessed a few emergent lateral roots. Due to the large seed size and slow rate of growth, peanut plants were not well suited for growing in pouches.White clover seeds (Trifolium repens L. cv. Regal Ladino) were obtained from Cal-West Seed Company, Woodland, CA. Seeds were surface-sterilized for 10 min with 5% sodium hypochlorite containing a drop of Tween 20 (Sigma Chemical Co.), washed, and then soaked in sterile H20 for 24 h in the cold (7). Swollen seeds were transferred to plates containing sterile half-strength Nfree Jensen's medium (10) with 0.5 g agar/L. Germinated seeds were transferred 24 h later to growth pouches wetted with 8 ml of the N-free Jensen's medium. Plants were inoculated 48 h after transfer to pouches with 100 id inoculum (A620nm = 0.08).In some cases, clover seedlings were grown on agar plates. The seeds were surface-sterilized, soaked in cold water, and germinated on Petri plates containing Fahraeus soft agar (10), 0.5 g agar/L.
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