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Xian Shen*, Ling Guo, and Zhenlin Wei

Malus hupenensis var. `Pingyitiancha' is an important apple stock with many good characteristics, including waterloggig resistance, cold resistance, salt resistance and so on. The three group gene-HVA1 come from barley was transformed into `Pingyitiancha' mediated by Agrobacterium tumefaciens and transformed regeneration plants were obtained in this research. The HAV1 gene cloned from plasmid containing it (offered by Dr. Guo Weidong) by PCR with high fidelity pfu Taq DNA polymerase. It was ligated between BamH 1 and Sac 1 site in PUC118 vector, and identified by electrophoresis after digested with BamH 1 and Sac 1. Through nuclear sequence detecting, it is confirmed that the HAV1 gene cloned in this research is 703bp.This fragment was ligated with 11kb fragment from pB121 plasmid and constructed pBHA vetor. The pBHA vector was introduced in A.tum LBA4404 by triparental mating and the binary vector was obtained. It is cinfirmed that HVA1 gene had been insert in T-DNA by in situ hybirdization. Using `Pingyitiancha' shoot apex, mediated by A. tum. System, the HAV 1 gene was transformed into the plant. Kam resistance regeneration plants were obtained, 6 of them were confirmed as transformation plants by PCR and dot blot.

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Jose A. Yuri, Claudia Moggia, Carolina A. Torres, Alvaro Sepulveda, Valeria Lepe, and Jose L. Vasquez

locations in Chile. Materials and Methods Plant material. A set of seven apple ( Malus ×domestica Borkh.) cultivars (‘Galaxy’, ‘Brookfield ® Gala’, ‘Super Chief’, ‘Fuji Raku Raku’, ‘Braeburn’, ‘Granny Smith’, and ‘Cripp's Pink’) grafted on two virus

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Mehdi Sharifi, Julia Reekie, Andrew Hammermeister, Mohammed Zahidul Alam, and Taylor MacKey

performance in an organic apple ( Malus domestica Borkh) orchard in northern Patagonia Plant Soil 292 193 203 Schmid, A. Weibel, F. 2000 Das Sandwich-System–ein Verfahren zur herbizidfreien Baumstreifenbewirtschaftung? [The Sandwich System, a procedure for

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Amy K. Szewc-McFadden, Warren F. Lamboy, and James R. McFerson

To comprehend genetic identity and relatedness in Malus germplasm held in situ and ex situ, we are employing simple sequence repeat (SSR) DNA fragment information in combination with passport and horticultural data. SSRs offer certain advantages for characterizing large arrays of germplasm efficiently. They are abundantly dispersed throughout plant genomes and are exceedingly polymorphic. In addition, they can be PCR-amplified and detected by automated fluorescence-based technology. A size-fractionated DNA library of M. ×domestica cv Golden Delicious was screened to identify SSR loci. Eight loci were found to be reliably informative and were used to prepare locus-specific primer pairs. Characterization of the 75 M. ×domestica accessions included in the core subset of the USDA-ARS Malus germplasm collection revealed six of the eight loci were polymorphic within the array. The number of alleles per locus ranged from two to 21. Throughput was enhanced by multiplexing, allowing simultaneous use of two or three primer pairs. With improved genetic characterization of Malus germplasm, we intend to better develop and relate the core subset to the rest of the collection and to in situ Malus genetic resources. SSR markers appear to be an efficient and reliable tool to expedite this process.

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Sun Qigao-

Through author's two years' study, mechanism of vitrification of Malus honanensis was conducted in following aspects:

  1. Factors affecting vitrification;
  2. Anatomical comparison of abnormal leaves and stems with those of the normal;
  3. Content6 of chlorophyll (a.b/T);
  4. contents of lignin, cellulose, etc;
  5. Contents of amino acid, protein;
  6. Isoenzyme of peroxidase, etc;
  7. Recovery of vitrious plantlets.

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D.K. Isutsa, I.A. Merwin, and B.B. Brodie

Apple replant disease (ARD) is a serious problem in fruit production, and none of the major clonal rootstocks are resistant to ARD. We have screened Malus domestica clones and species accessions from the USDA Malus Germplasm Repository at Geneva, N.Y., including M. angustifolia-2375.03 (MA), M. coronaria-2966.01 (MC), M. fusca-3031.01 (MF), M. ioensis-3059.01 (MI), M. sieversii-3530.01 (MS), and M. kirghisorum-3578.01 (MK), for resistance to ARD and root-lesion nematodes (RLN, Pratylenchus spp.), in a composite soil collected from 11 New York orchards with known ARD. Plant dry mass and height, root necrosis, and nematode populations in different apple species and clones were compared after 60 days growth in steam-pasteurized (PS), RLN-inoculated (IS), and naturally infested field (FS) soils with 1200 RLN per 100 cm3. More severe stunting, reduced plant dry mass, and root necrosis occurred in FS seedlings compared with those in PS, but M. angustifolia seedlings were substantially more resistant or tolerant to RLN and ARD than the other species tested. Plant dry mass ranked MK>MS>MA>MI>MF>MC, and these differences were significant at the 5% level. RLN root populations were negatively correlated with plant dry mass, and accounted for about 10% of its variation, with nematode populations in roots ranking MC>MF>MK>MI>MS>MA. Useful resistance to ARD and parasitic nematodes apparently exists within Malus germplasm collections, and can be identified by testing more genotypes, developing rapid resistance screening methods, and comparing RLN host preferences among Malus genotypes and various orchard cover crops.

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Laura L. Benson, Warren F. Lamboy, and Richard H. Zimmerman

The U.S. National Plant Germplasm System (NPGS) currently holds 36 separate accessions of the `Yichang' clone of Malus hupehensis (Pamp.) Rehd. The `Yichang' clone originally entered the United States in 1908 as seed collected for the Arnold Arboretum by E.H. Wilson near Yichang, Hubei Province, China. The original description of M. hupehensis omits fruit characters, and botanists frequently augment these omissions with descriptions of the `Yichang' clone. Apomixis occurs in Malus, including M. hupehensis, and is strongly associated with elevated ploidy levels. Simple sequence repeats (SSRs) were used to characterize 65 accessions of M. hupehensis. To check for polyploidy, a set of M. hupehensis accessions was evaluated with flow cytometry. The simple sequence repeat phenotypes and ploidy information revealed the `Yichang' clone under various accession names in arboreta. It was neither known nor suspected that the U.S. National Plant Germplasm System held many duplicate accessions of the `Yichang' clone prior to their molecular characterization. Germplasm conservation decisions for Malus species can benefit from an increased knowledge of the genetic variation or lack thereof in naturalized populations and ex situ collections.

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Laura L. Benson, Warren F. Lamboy, and Richard H. Zimmerman

Simple sequence repeats (SSRs) are highly polymorphic regions of DNA that can be used for the molecular characterization of apple (Malus) germplasm. SSR markers are sufficiently variable to distinguish between individual plants in wild Malus species. In this study, accessions of Malus hupehensis were screened for fragment length variation in PCR amplified simple sequence repeat regions of DNA. The fragment length phenotype produced by five SSR primer pairs showed no variation between two lineages of M. hupehensis collected in the Changjiang (Yangtse) River valley. One lineage was collected by E.H. Wilson in 1908 near the city of Ichang, Hubei Province. The second lineage was collected by cooperators at China's Southwest Agricultural University (SWAU) in 1997 near the city of Chongqing (Chungking). M. hupehensis Plant Introduction No. 588760 from the National Plant Germplasm System lacks provenance, but displays a fragment length phenotype identical to both the Wilson and SWAU lineages. The spread of a clone may be aided by asexual reproduction through seed, which is not uncommon in polyploid apples. Two seedlings each of 15 maternal trees from the SWAU lineage were assayed for ploidy level by flow cytometry. The DNA content per nucleus for all SWAU progeny fell within the range for triploids, 2.19 to 2.68 pg DNA/nucleus. It appears that plant explorers in China separated by almost 90 years have succeeded in sampling a single clonal lineage of M. hupehensis.

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Stan C. Hokanson, Amy K. Szewc-McFadden, Warren F. Lamboy, and James R. McFerson

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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Roberto Hauagge and James N. Cummins

Dormancy patterns throughout the season were studied in more than 90 apple (Malus ×domestica Borkh.) cultivars and related Malus spp. The seasonal apple bud dormancy pattern resembles a normal curve: it starts to intensify soon after bud formation and reaches maximum intensity by the time of leaf fall/senescence. Genotypes were grouped into three general classes based on maximum dormancy intensity. Maximum intensity of bud dormancy measured in cold winters is inversely related to adaptation to the subtropics. Low-chilling requirement (CR) cultivars have a shallow depth of dormancy with very little alteration throughout the year. High-CR cultivars have intense bud dormancy, the first stage of which can be induced by growing these cultivars at temperatures above 20C. Genotypes differed in their rates of dormancy dissipation. The efficiency of chilling unit (CU) accumulation to break dormancy was negatively correlated with CR, which indicates the importance of factors other than CU accumulation in terminating bud dormancy in low-CR cultivars. The inherent length of bud dormancy plays a major role in determining the time of budbreak in the spring. Deviations may be related to the genotypic efficiency in which chilling modifies dormancy and possibly the basal temperatures to which buds respond. Chill unit requirement and heat unit requirement are dependent factors. Heat requirement comparisons may be meaningless if the dormancy intensities of the genotypes are not taken into consideration.