SUPPLEMENTARY MATERIAL
Exploration of genetic diversity among medicinally important genus Epimedium species
based on genomic and EST-SSR marker
Zubaida Yousaf a, c Weiming Hub, Yanjun Zhangb, Shaohua Zenga and Ying Wang b, a
a: Key Laboratory of Plant Resources Conservation and Sustainable utilization, South China
Botanical Garden, Chinese Academy of Sciences, Gaungzhou 510650, PR China
b: Key Laboratory of Plant Germplasm Enhancement and Speciality Agriculture, Wuhan
Botanical Garden, the Chinese Academy of Sciences, Wuhan, Hubei, China
c: Department of Botany, Lahore College for Women University, Jail Road Lahore, Pakistan.
03334272550, zubaida_yousaf@yahoo.com
Abstract: Epimedium species has gained prime importance due to their medicinal and economic
values. Therefore in the present study, 26 genomic SSR and 10 EST-SSR markers were
developed for 13 medicinal species of the Epimedium genus and one out group species
Vancouveria hexandra W. J. Hooker to explore the existing genetic diversity. A total of 100
alleles by genomic SSR and 65 by EST-SSR were detected. The genomic SSR markers were
presented between 2-7 alleles per locus. The observed heterozygosity (Ho) and expected
heterozygosity (He) were ranged from 0.00 to 4.5 and 0.0254-2.8108 respectively. Similarly, for
EST-SSR, these values were ranged from 3.00-4.00 and 1.9650- 2.7142. The number of alleles
for EST-SSR markers ranged from 3-10 with an average of 3.51 per loci. It has been concluded
that medicinally important species of the genus Epimedium possesses lower Intraspecific genetic
variation.
Key words: Epimedium, Genomic SSR/EST SSR markers, Genetic diversity, Systematics
Experimental:
A total of 13 medicinally important species of the genus Epimedium including 45
individuals (Collected from the different phytogeographical regions of China (Fig S5)) were
selected for the taxonomical evaluation based on genomic and EST-SSR markers. According
to the classification of Stearn 2002, sampled species covered two sections (Diphyllon and
Macroeras) and two series (Brachycerae and Dolichocerae) of section Diphyllon (Fig. S1).
Vancouveria hexandra W. J. Hooker was used as out group species due to its close
relationship to the genus Epimedium (Wang et al. 2007).
In the present study, 26 genomic SSR and 10 EST SSR primers were randomly selected
from already generated libraries in Key Laboratory of Plant Resources Conservation and
Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences,
Gaungzhou, China. PCR amplification was performed in a total volume of 10μl reaction
mixture containing 50ng template DNA, 20 pmol of each primer, 10×buffer [10 mM TrisHCl (pH 8.4), (1-2mM) MgCl2, 200μM NTPs and 0.5 unit of Taq DNA polymerase. The
concentration of MgCl2 is locus specific. The PCR reactions were performed under standard
conditions for all primers in lab cycler (PCR machine). The annealing temperature was fixed
for all primer pairs at 55˚C. After 5 minutes at 95˚C, 34 cycles were carried out with 30s at
95˚C, 30s at 55˚C, 90s at 72˚C for extension, and final extension step of 10 minutes at 72˚C.
The separation of alleles was performed on 6% polyacrylamide gel. PCR products were
mixed with 7.5μl of loading buffer. The mixture was denatured at 94˚C for 10 min before
loading onto the gels. Gels (20 bp marker, by Beijing Yuanpinghao Biotech Co., Ltd) were
stained with silver nitrate following the protocol as described in Bassam et al. (1991) that
was used for calculating the length of genomic and EST-SSR amplicons. Preliminary
population genetics analyses were performed using GENEPOP version 3.4 (Raymond and
Rousset, 1995). Cluster analysis based on Nei genetic distance was performed using GenAlex
6 combination with Mega 4.
Fig S1: lower order taxonomy of the genus Epimedium followed by Stearn, 2002,
Fig S2: Cluster analysis of medicinally important species of the genus Epimedium based on
genomic SSR markers
Fig S3: Cluster analysis of medicinally important species of the genus Epimedium based on ESTSSR markers
Fig S4: Cluster analysis of medicinally important species of the genus Epimedium based on
Twenty six genomic and ten EST- SSR markers
Fig S5: Phytogeographical presentation of species of the genus Epimedium in China
Table S1: List of Germplasm Epimedium species with their accession used for genetic diversity exploration
Serial
#
Individuals
species
(Accession numbers)
1.
E. pubescens
SCI-1, SCI-2, SCI-3, SCI-4
2.
E. sagittatom
HNI-11, GDI-6,FJI-1, LTI-3
3.
E. accuminatum
SCYI-2, SCYI-3, SCYI-5, SCYI-8
4.
E. brevicornum
SFI-23, HNSSI-39, 903, 904
5.
E. koreanum
CY2-27, CY1015, CY1017, CY1018
6.
E. wushanense
WY3-6, WY3-10, WY245, WY425
7.
E. leptorrhizum
LC2-4, LC2-7, LC2-27, LC2-30
8.
E. brachyrhizum
HN4
9.
E. membranace
SCLI-5, SCLI-7, SCLI-8, SCLI-9
10.
E. rhizomatosum
34-38
11.
E. chlorandrum
SCy3-7, SCy3-8, SCy3-14, SCy3-15
12.
E. diphyllum
39.84, 40.105
13.
E. stelluatumI
S*2-27, S*2-37, S*2-38, S*2-42
14.
Vancouveria hexandra
45 S 81