The Enzyme mix

Percentage Enzymes Make

0.7 Cellulase R10 Duchefa C8001
0.7 Cellulase CalBioChem 319466
1 Pectolyase Sigma P3026
1 Cytohelicase Sigma C8274
Differences in the Patterns of a Microsatellite Marker on Chromosomes in Four Allium Species

Bulbs and seeds of A. ampeloprasum L., A. ascalonicum L., A. cepa L. and A. sativum L. were collected
from botanical gardens and agricultural areas in provinces of Thailand and then planted and
germinated at the Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen,
Thailand. The root tips were excised and kept in cold water for 30 min for the preparation of
metaphase chromosomes. In some cases, before root tips were collected, kinetin (4 mg mL-1 in
water) was applied to the plant. The plant was placed outdoors or illuminated by a 25 W lamp for 30
min to stimulate cell division. The roots were excised at 9:30–11:00 a.m. Root tips in cold water were
transferred to 0.05% colchicine solution for 24 h and then were fixed in ethanol– acetic acid of 3 : 1
(v/v) at 4°C for 24 h. The samples were transferred to 90% ethanol for storage at 4°C

Thereafter, the tips were treated with a solution of 2.5% cellulase (Sigma) and 2.5% pectinase
(Sigma) at 37°C for 90–120 min and then hydrolyzed in 5% HCl at 60°C for 10 min. The root tips were
broken with a dissecting needle in 60% acetic acid in a tube and the tube was tapped to resuspend
the cells. The cell suspension was dispensed onto a slide and squashed with a coverslip. The slide was
immersed in liquid nitrogen to remove the coverslip and subsequently air dried at room temperature
for 24 h
Comparative Tyramide-FISH mapping of the genes controlling flavor and bulb color in Allium species
revealed an altered gene order

Chromosome preparation

Seeds of A. cepa. L., var. ‘Haltsedon’ (2n = 2x = 16), A. fustulosum L., var.‘Russkiy Zimniy’
(2n = 2x = 16), A. schoenoprasum L., var. ‘Medonos’ (2n = 2x = 16), A. nutans L. (2n = 4x = 32) and A.
altaicum Pall., var. ‘Alves’ (2n = 2x = 16) were germinated on moist filter paper at 25 °C. Seedlings
with roots about 1 cm long were pre-treated in 0.75 mM hydroxyurea for 20 h at RT and then in
0.05% colchicine for 3.5–4 h at RT. Plants of A. oschaninii O. Fedtsch(2n = 2x = 16), A. pskemense O.
Fedtsch (2n = 2x = 16) and A. roylei Stearn (2n = 2x = 16) were grown in pots in a greenhouse. In order
to arrest chromosomes at the metaphase stage, young roots were treated in a nitrous oxide gas
chamber at elevated pressure (1.0 MPa) for 2 h and then were submerged in a saturated aqueous
solution of α-bromonaphthalene (1:1000, v/v) overnight at 4 °C. The root tips were fixed in freshly
prepared 3:1 (v/v) ethanol:acetic acid mixture for 1 h at RT and stored at −20 °C. Chromosome
preparations were made according to the SteamDrop protocol59 using a cell suspension obtained
after digestion of root tips in 0.1% enzyme mix (Pectolyase Y-23, Kikkoman, Tokyo, Japan; Cellulase
Onozuka R-10,Yakult Co. Ltd., Tokyo, Japan and Cytohelicase, Sigma-Aldish Co.LLC, France) for 90–
100 min at 37 °C.
Cenh3 ALIIUM

Root tips of A. fistulosum were pretreated at 0°C for 20 h in water. Pretreated root tips were fixed in
ethanol/glacial acetic acid (3∶1), and then dividing cells in the root tips were squeezed in 45% acetic
acid on slide glasses