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  • Author or Editor: Warren F. Lamboy x
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The U.S. Department of Agriculture (USDA), Agricultural Research Service (ARS) tetraploid cherry (Prunus L. sp.) collection at Geneva, N.Y., contains ≈75 accessions of sour cherry (P. cerasus L.), ground cherry (P. fruticosa Pall.), and their hybrids. Accurate and unambiguous identification of these accessions is essential for germplasm preservation and use. Simple sequence repeats (SSRs) are currently the markers of choice for germplasm fingerprinting because they characteristically display high levels of polymorphism. Recently SSR primer pairs from sweet cherry (P. avium L.), sour cherry, and peach [(P. persica L. Batsch (Peach Group)] have been reported. Ten SSR primer pairs were tested on 59 tetraploid cherry accessions to determine if they could differentiate among the accessions. Scorable SSR fragments were produced with all primer-accession combinations. The cherry accessions exhibited high levels of polymorphism with 4 to 16 different putative alleles amplified per primer pair. Most of the putative alleles were rare with frequencies <0.05. Heterozygosity values ranged from 0.679 to 1.00, while gene diversity values ranged from 0.655 to 0.906. The primer pairs differentiated all but two of the 59 cherry accessions. Based upon the ability of the SSR data to differentiate the cherry accessions and the high level of gene diversity, we propose that all the tetraploid cherry accessions in the USDA/ARS collection be fingerprinted to provide a mechanism to verify the identity of the individual accessions. The fingerprinting data are available on the World Wide Web (http://www.ars-grin.gov/gen/cherry.html) so that other curators and scientists working with cherry can verify identities and novel types in their collections and contribute to a global database.

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A diverse collection of 133 Malus species and hybrids from the USDA Plant Genetic Resources Unit's core subset collection was screened with five simple sequence repeat (SSR) primer pairs in order to determine genetic identities and overall levels of genetic variation. The number of amplification products (alleles) per locus (primer pair) in this collection ranged from 6 to 39, with some genotypes showing complex banding patterns of up to four products per locus, suggesting that duplication events may have occurred within the genome. Five primer sets unequivocally differentiated all but 10 pairs of genotypes in the collection, with seven of these 10 being pairs of the same species. Within three of the species holdings surveyed, M. honanensis, M. sargentii, and M. sikkimensis, no genetic variation was revealed with the SSR markers. The discrimination power for the combined loci in this collection was nearly one, which indicates that the likelihood of two genetically different accessions sharing the same alleles at all the loci included in this study would be nearly impossible. Coupled with results from a previous survey of M. × domestica accessions, this finding suggests that with five SSR primer pairs, the majority of the Malus holdings could be assigned a unique fingerprint identity. The average direct count heterozygosity over all loci was 0.620, ranging in value from 0.293 to 0.871 over individual loci. These heterozygosity counts will be compared with a survey of naturally occurring M. sieversii to determine whether current repository holdings are representative of the overall levels of diversity occurring in Malus. Information generated with this study, coupled with passport and horticultural data will inform curatorial decisions regarding deaccessioning of duplicate holdings and plans for future germplasm collections.

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The USDA–ARS germplasm collection of cold-hardy Vitis held at the Plant Genetic Resources Unit, Geneva, N.Y., has over 1300 clonal accessions maintained as field-grown vines. Security back-up using field-grown or potted vines at remote sites or via in vitro methods is costly. Cryopreservation offers a safe, cost-effective alternative. While we routinely employ cryogenic storage of dormant buds of Malus, dormant buds of Vitis generally do not appear to tolerate the desiccation levels required by our current cryopreservation protocol. Since tolerance to desiccation and cold appear to be correlated in Vitis, we tested desiccation tolerance of 60 germplasm accessions selected from the core subset to represent a range of cold hardiness. Budwood was collected in December 1995 in Geneva, stored at –4°C in sealed bags, and systematically desiccated to 30% and 20% moisture. In some treatments, additional desiccation was imposed by slow freezing to –25°C. Microscopic examination of rehydrated buds indicated 60% of accessions tolerated desiccation as low as 20% moisture. Freeze-desiccation at –25°C after desiccation at –4°C neither increased nor decreased viability in these accessions. Only slight modification so current protocols should be necessary for cryopreservation of this class. Of the remaining accessions, 25% tolerated desiccation to 30% moisture, but 15% were intolerant to any desiccation level tested. Techniques must be developed to successfully cryopreserve both these classes of accessions.

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Fifty-two germplasm accessions of Chinese vegetable Brassicas were analyzed using 112 random amplified polymorphic DNA (RAPD) markers. The array of material examined spanned a wide range of morphological, geographic, and genetic diversity, and included 30 accessions of Brassica rapa (Chinese cabbage, pakchoi, turnip, broccoletto), 18 accessions of B. juncea (leaf, stem, and root mustards), and 4 accessions of B. oleracea ssp.alboglabra (Chinese kale). The RAPD markers unambiguously identified all 52 accessions. Net and Li genetic similarities were computed and used in UPGMA cluster analyses. Accessions and subspecies clustered into groups corresponding to the three species, but some accessions of some subspecies were most closely related to accessions belonging to another subspecies. Using genetic similarities, it was found that Chinese cabbage is more. likely to have been produced by hybridization of turnip and pakchoi, than as a selection from either turnip or pakchoi alone. RAPD markers provide a fast, efficient technique for diversity assessment that complements methods currently in use in genetic resources collections.

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Fifty-two germplasm accessions of Chinese vegetable brassicas were analyzed using 112 random amplified polymorphic DNA (RAPD) markers. The array of material examined spanned a wide range of morphological, geographic, and genetic diversity, and included 30 accessions of Brassica rapa L. (Chinese cabbage, pakchoi, turnip, and broccoletto), 18 accessions of B. juncea (L.) Czern. (leaf, stem, and root mustards), and four accessions of B. oleracea L. ssp. alboglabra (Chinese kale). The RAPD markers unambiguously identified all 52 accessions. Nei-Li similarities were computed and used in unweighed pair group method using arithmetic means (UPGMA) cluster analyses. Accessions and subspecies were clustered into groups corresponding to the three species, but some accessions of some subspecies were most closely related to accessions belonging to other subspecies. Values for Nei-Li similarities suggest that Chinese cabbage is more likely to have been produced by hybridization of turnip and pakchoi than as a selection from either turnip or pakchoi alone. RAPD markers are a fast, efficient method for diversity assessment in Chinese vegetable brassicas that complements techniques currently in use in genetic resources collections.

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Malus sieversii, the main progenitor of domesticated apple, is native to areas in Central Asia. To better represent Malus wild germplasm in the USDA–ARS germplasm collections, maintained in Geneva, N.Y., a cooperative project was initiated with the Republic if Kazakhstan to collect and assess that country's wild populations of M. sieversii and to develop more secure in situ reserves to complement ex situ holdings in the United States and Kazakhstan. To date, four exploration trips to the region have included participants from the United States, Kazakhstan, Canada, New Zealand, and South Africa. Four Kazkh scientists have toured USDA–ARS sites, exchanged information, and collected germplasm in the United States greenhouse screens of 1600 have revealed potentially new sources of resistance to apple scab, cedar apple rust, and fire blight. An isozyme analysis of maternal half-sib families from four regions suggests the populations of M. sieversii collected represent a single panmictic population, with over 85% of total genetic variation due to differences among families. The most recent collections in 1995 were directed towards more ecologically diverse regions, including a site (Tarbagatai) at the most northern limit for M. sieversii equivalent to northern Minnesota in the United States. Some trees in this region produced fruit nearly 70 mm in diameter with excellent aroma, firmness, and color. This germplasm is being systematically characterized for horticultural traits, pest and disease resistance, and molecular markers.

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Clonally propagated crops, unlike seed-propagated crops, require intense and costly maintenance, generally in ex situ field gene banks. Consequently, large germplasm collections of tree species especially, are difficult to conserve in a well-replicated fashion and are vulnerable to damage from environmental stresses. Accordingly, long-term storage in liquid nitrogen presents a viable conservation alternative. To assess effectiveness of one approach to cryopreservation, dormant buds from 64 apple (Malus ×domestica Borkh. and other Malus spp.) accessions were collected and preserved in liquid nitrogen using a dormant-vegetative-bud method. Buds were retrieved from liquid nitrogen storage, rehydrated, and grafted onto rootstocks to determine survival. Mean recovery was 76% for 40 cold-hardy accessions, 66% for 20 moderately cold-hardy accessions, and 24% for four cold-tender accessions (range: 16% to 100%). Only four accessions had ≤25% recovery while 54 accessions had ≤50% recovery and 35 accessions had ≤75% recovery. No significant decline in recovery of these accessions by bud grafting occurred after 4 years of liquid nitrogen storage.

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