Marker details for GD162

Phil Forsline and his collaborators brought back Malus sieversii seeds and clones from several collection sites in Kazakhstan in 1995 and 1996. These analyses compare the genetic diversity of 274 M.sieversii individuals from collection site 9 and 174 individuals of M. sieversii from collection site 6. The purpose of the study was to compare allelic diversity with phenotypic diversity and identify core subsets of individuals that capture much of the characterized diversity from these 2 collection sites. This paper has been published (see Evaluation citation).

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Leaves of 2 samples of each individual were extracted using DNeasy 96 plant kits (Qiagen). Malus SSRs were amplified with forward primers labeled with either IRD 700 or IRD800 obtained from MWG-Biotech. Unlabeled reverse primers were purchased from IDT (Coralville, Iowa). PCR was carried out in 15 uL total volume. 10-50 ng DNA template and primers (0.3 pM) were combined with 1.5 units Taq polymerase (Promega), 1x Promega Magnesium free buffer (10 mM Tris-HCl, 50 mM KCl, and 0.1% Triton X-100), 0.25 mM MgCl2, adn 0.25 mM dNTP. PCR amplifications were carried out using MJ Research PTC200 or Dyad Thermocyclers. A touch-down program reduced the annealing temperature 2 C every other cycle starting at 63C and ending at 57C, followed by an annealing temperature of 55C for 20 additional cycles, and ended with a 2 min 72C extension. Completed PCR reactins were diluted 1:1 in 95% formamide, 50 mM EDTA, bormophenol blue loading dye, and denatured at 95C for 3 min. Gels (6.5% LI-COR KB Plus acrylamide) were run in 1x TBE (89 mM Tris, 89 mM boric acid, 20 mM EDTA) buffer for 1 h 45 min at 1500 V, 40 W, 40 mA, and 45C on a LI-COR 4200 DNA sequencer.

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Digital images were collected from the sequencer using LI-COR Saga Generation 2 software and were manually analyzed using the Saga software. Alleles from replicate samples were examined at each locus and when alleles for replicates were not identical, data for that locus were entered as missing in subsequent analyses.

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PI 590184:214,234; PI 588853:214,214; PI 588850:226,238

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Seven microsatellites were used to analyze genetic diversity of 496 Malus orientalis accessions grown from seeds collected in Russia (1998) and Turkey (1999). Results have been published (Volk et al., (2008) J. Am. Soc.Hort. Sci.

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Leaves of 2 samples of each individual were extracted using DNeasy 96 plant kits (Qiagen). Malus SSRs were amplified with forward primers labeled with either IRD 700 or IRD800 obtained from MWG-Biotech. Unlabeled reverse primers were purchased from IDT (Coralville, Iowa). PCR was carried out in 15 uL total volume. 10-50 ng DNA template and primers (0.5 pM) were combined with 1.5 units Taq polymerase (Promega), 1x Promega Magnesium free buffer (10 mM Tris-HCl, 50 mM KCl, and 0.1% Triton X-100), 0.25 mM MgCl2, adn 0.25 mM dNTP. PCR amplifications were carried out using MJ Research PTC200 or Dyad Thermocyclers. A touch-down program reduced the annealing temperature 2 C every other cycle starting at 63C and ending at 57C, followed by an annealing temperature of 55C for 20 additional cycles, and ended with a 2 min 72C extension. Completed PCR reactins were diluted 1:1 in 95% formamide, 50 mM EDTA, bormophenol blue loading dye, and denatured at 95C for 3 min. Gels (6.5% LI-COR KB Plus acrylamide) were run in 1x TBE (89 mM Tris, 89 mM boric acid, 20 mM EDTA) buffer for 1 h 45 min at 1500 V, 40 W, 40 mA, and 45C on a LI-COR 4200 DNA sequencer.

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Digital images were collceted from the sequencer using LI-COR Saga Generation 2 software and were manually analyzed using the Saga software. Alleles from replicate samples were examined at each locus and when alleles for replicates were not identical, data for that locus were entered as missing in subsequent analyses.

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PI 588853 (Coxs Orange Pippin):214,214; PI 590184 (Golden Delicious):214,234; PI 588850 (Rome Beauty Law):226, 238

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2

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