Evaluation location: North Dakota, United States
To analyze chromosome number and ploidy level of the Thinopyrum accessions, we prepared chromosome spreads following the procedures of Kato et al. (2006) and Han and Lv (2013) with some minor modification. The seeds were germinated on moist filter paper at 23 degrees C for 48 hours. Root tips of 1 to 2 cm length were collected and pretreated with nitrous oxide (N2O) at 10 atm pressures for 2 h in a gas chamber, as described by Kato et al. (2006). The root tips were then fixed in 90% acetic acid for 10 min and washed three times with distilled water. The apical meristem of the root tips were collected in 0.5 mL microcentrifuge tube and treated with a mixture of 1% pectinase (Yakult Pharmaceutical, Tokyo, Japan) and 2% cellulase (Yakult Pharmaceutical, Tokyo, Japan) for 45 to 50 min. Then the root sections were washed three times with 75% ethanol and crushed by steel needles to form a suspension solution of single cells. After centrifugation at 4000 x g for 3 min, the cell pellet was washed with 75% ethanol and resuspended in 100% acetic acid. About 8 uL of cell suspension solutions were dropped onto a clean slide. After the slide was air dried, the root cells were examined under an Olympus BX53 phase contrast microscope. A minimum of 15 metaphase cells were observed for each accession. On the basis of chromosome number, accessions were classified as diploid (2x), tetraploid (4x), hexaploid (6x), octoploid (8x), nonuploid (9x), or decaploid (10x). Accessions expressing aneuploidy were classified on the basis of clustering of chromosome number around 2n = 42, 56, 63, or 70.
Excel file with chromosome numbersdocx file of Seeding reactions of the five Thinopyrum species to nine races of Puccinia graminis f. sp. triticidocx file of marker genotypes