Plant Genome Size and Ploid Analysis
Material：Plant leaf tissue
Nuclei DNA standard：
chicken erythrocyte nuclei, CEN 2.5pg/2C
trout erythrocyte nuclei, TEN 5.5pg/2C
triploid trout nuclei, TTN 8.25pg/2C
G buffer
PI stock：1mg/ml
RNase (DNase free) stock：20mg/ml
Ice bucket
Petri dish
Shave blade
Falcon tube
Method：
1. add 0.2g leaf tissue into 1ml G-buffer (added 0.5% 2- mercaptoethanol in advance)
and mince them with shave blade, then filtrate them with 30um nylon membrane
into Falcon tube, keep the tube on ice.
2. add dye (50ug/ml for PI) into the tube, mix them gently and stand on ice for 1520 minutes.
** You can also add RNase（final concentration 10ug/ml） and keep the tube in
37℃ for 30 mins to digest RNA.
3. analyze DNA standard at first to verify instrument condition and detector setting,
then analyze samples. Record peak mean in PI histogram.
4. estimate samples genome size as below:
sample genome size (pg)=
( sample peak mean/ standard peak mean)x standard genome size (pg)
5. Refer to ploid analysis, take diploid sample as your standard and analyze it first,
compare standard and sample peak mean to know sample ploid. For example,
peak mean of diploid is 300 and sample peak mean is 450 or so, it means the
sample is tetraploidy.
※ Some plants can be taken as genome size standards, just extract nuclei from
standard plant as from your sample plants then analyze them.
※ Plant genome size standard:
Source：Annals of Botany 95: 99~ 110, 2005
Examples:
(1) genome size estimating
The left is CEN peak and the right peak comes
from sample, the sample genome size is: 417.3
(sample peak mean) / 355.1 (CEN peak mean) x
2.5 (CEN genome size) = 2.94pg
(2) ploid analysis
Peak A comes from diploid standard and peak B,
C are unknown samples. By comparing peak
mean of A, B, C, we can know the sample B is
tetraploid（531.8/ 274.8= 1.94）and sample C is
hexaploid（781.4/ 274.8= 2.84）.