Genetic diversity analysis of yams (Dioscorea spp.) cultivated in China using ISSR and SRAP markers

RESEARCH ARTICLE

Genetic diversity analysis of yams (Dioscorea spp.)cultivated in China using ISSR and SRAP markers

Zhi Gang Wu • Xiao Xia Li • Xin Chun Lin •

Wu Jiang • Zheng Ming Tao • Nitin Mantri •

Chuan Yin Fan • Xiao Qing Bao

Received: 17 May 2013 / Accepted: 26 November 2013 / Published online: 22 January 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Yam (Dioscorea spp.) is widely cultivated

in China and many landraces are maintained by local

farmers. However, there is little information available

about their diversity and species identity. In this study,

inter simple sequence repeat (ISSR) and sequence

related amplified polymorphism (SRAP) techniques

were used to assess genetic diversity within 21 yam

landraces from seven cultivated populations. We

observed high level of polymorphism among these

landraces, specifically, 95.3 % for ISSR and 93.5 % for

SRAP. Analysis of molecular variance revealed a

significantly greater variation among the four yam

species (40.39 %) and their populations (35.78 %) than

within the populations (23.83 %). The unweighted pair

group method arithmetic averages clusters and principal

component analysis for 21 landraces formed four well-

separated groups containing landraces of each of the four

species, namely, Dioscorea opposita Thunb., Dioscorea

alata L., Dioscorea persimilis Prain et Burkill, and

Dioscorea fordii Prain et Burkill. The ISSR and SRAP

primers were highly discriminatory among the 21

landraces; all 21 landraces could be easily differentiated

using these primers. The average mean of gene flow

(Nm = 0.1081) estimated from high genetic differenti-

ation (Gst = 0.8222) suggested that gene flow among

the populations was relatively restricted. The lack of

genetic diversity within individual yam species suggests

that it is critical to develop long-term strategies for

enhancing genetic diversity within various yam species.

Keywords China � Dioscorea � Genetic

diversity � ISSR � SRAP � Yam

Electronic supplementary material The online version ofthis article (doi:10.1007/s10722-013-0065-2) contains supple-mentary material, which is available to authorized users.

Z. G. Wu (&) � X. X. Li � W. Jiang � Z. M. Tao (&) �C. Y. Fan � X. Q. Bao

Zhejiang Institute of the Subtropical Crops, No. 334

Xueshan Road, Wenzhou 325005, Zhejiang Province,

People’s Republic of China

e-mail: [email protected]

Z. M. Tao

e-mail: [email protected]

X. X. Li � C. Y. Fan � X. Q. Bao

Wenzhou Medical College, Wenzhou 325035, People’s

Republic of China

X. C. Lin

Key Lab for Modern Silvicultural Technology of

Zhejiang, Zhejiang Forestry University, Linan 311300,

People’s Republic of China

N. Mantri

School of Applied Sciences, Health Innovations Research

Institute, RMIT University, Melbourne, VIC, Australia

123

Genet Resour Crop Evol (2014) 61:639–650

DOI 10.1007/s10722-013-0065-2

http://dx.doi.org/10.1007/s10722-013-0065-2

Introduction

Yams belong to the genus Dioscorea and family

Dioscoreaceae. They are an important food crop in

Southeast Asia, West Africa, tropical America, and

other subtropical regions (Burkill 1960; Coursey 1967).

The domestication of wild yams was a common practice

mainly in West Africa and East Asia and it offered an

insight into how farmers tap wild genetic resources to

create products suitable for agriculture. At least 50 of

more than 600 known species of Dioscorea have been

domesticated for food and medicinal use (Hahn 1995).

Some researches revealed that these domesticated

species can be divided into three cultivated taxon,

namely, Eastern Asia (basic chromosome number

X = 10), Africa (basic chromosome number X = 10)

and America (basic chromosome number X = 9) (Qin

et al. 1985; Rubatzky and Yamaguchi 1997). Yams have

been known to have considerable diversity both at inter-

and intra-specific levels (Okoli 1991). This diversity has

been exploited for ongoing domestication of wild yams

in tropical and subtropical countries (Dumont and

Vernier 2000; Scarcelli et al. 2006).

China is an important and isolated yam domestica-

tion center (Coursey 1967; Qin et al. 1985). Various

Dioscorea species have been domesticated and widely

cultivated as edible resources and medical materials. It

is believed that farmers collected the tubers of wild

yams and brought under cultivation with intense

vegetative multiplication and selection procedure that

induced morphological or biochemical changes,

mainly in tuber characteristics, making it a completely

different variety (Mignouna and Dansi 2003). As a

result, Chinese farmers have developed many varieties

with different phenotypic traits (e.g., tuber shape,

tuber flesh color, leaf traits) through the long-term

domestication process (Huang 2005). Most of the

varieties that are currently cultivated are accessions

selected by farmers from existing landraces. However,

until recently, only limited research has been done to

understand this process of domestication followed by

Chinese farmers to generate agricultural biodiversity.

The extent of genetic diversity in yam landraces and

their relationship are yet to be investigated in detail.

Not only yam diversity, but also the species

identification of Chinese yam is still obscure. In

particular, some yam species/landraces are known to

have medicinal properties and have been used in tra-

ditional Chinese medicine, but incorrect identification

of these species/landraces has led to ineffective

treatment (Xu and Xu 1997). In previous studies,

attempts to characterize yams using plant morphology

(Cai et al. 1999), pollen morphology (Shu 1987),

cytotaxonomy (Qin et al. 1985) and isozyme markers

(Xia et al. 2004) did not give robust results due to their

high degree of variability. Moreover, the narrow range

of morphological traits and the limited number of

polymorphic enzyme markers are not adequate to

discriminate between all yam landraces, and unable to

assess the level of genetic diversity (Cai et al. 1999;

Dansi et al. 2000b).

Progress has recently been made, thanks to the use

of molecular markers. Various molecular markers,

such as Random Amplified Polymorphic DNA

(RAPD), Inter Simple Sequence Repeat (ISSR),

Restriction Fragment Length Polymorphism (RFLP),

Amplified Fragment Length Polymorphism (AFLP),

and Microsatellites or Simple Sequence Repeats

(SSR), are increasingly used for yams taxonomic

classification, phylogenetics, genetic linkage map

construction, cultivar identification, and diversity

studies (Wilkin et al. 2005; Scarcelli et al. 2006;

Tamiru et al. 2007; Tostaina et al. 2007; Sartie et al.

2012; Nascimento et al. 2013). These molecular tools

enabled detection of differences among yam cultivars

that were considered to be similar based on morpho-

logical and isozyme markers, and demonstrated their

usefulness as discriminative tools in yams (Dansi et al.

2000a). However, little information is available on the

genetics of Chinese yams. To date, only two studies

have reported the genetic diversity in Chinese yams

using RAPD and ISSR markers (Zhou et al. 2005; Hua

et al. 2009). These two studies did not present

comprehensive species identification of yam land-

races. Moreover, RAPD markers have limited appli-

cability because of instability and poor reproducibility

(Budak et al. 2004; Bahieldin et al. 2006). A more

advanced method of DNA fingerprinting is the

Sequence Related Amplified Polymorphism (SRAP),

which aims at the amplification of open reading frames

(ORFs) and is simple, reproducible and has reasonable

throughput rate. ISSR and SRAP markers have been

shown to be effective for genetic diversity analysis,

species identification and germplasm evaluation (Li

and Quiros 2001; Budak et al. 2004). Meanwhile, the

use of multiple molecular methods can potentially

provide a more robust evaluation of diversity level than

a single method (Ramser et al. 1997; Shao et al. 2010).

640 Genet Resour Crop Evol (2014) 61:639–650

123

Previous studies (Zhou et al. 2005; Hua et al. 2009)

mostly evaluated cultivars or landraces from Diosco-

rea opposita Thunb., and used a single molecular

method for diversity analysis. Therefore, the main

objectives of this study was to use ISSR markers in

combination with SRAP markers for a detail analysis

of genetic diversity within yam landraces in China.

Further, we also determined the species of these

landraces by utilizing various genotypes representing

the main cultivated yam species as reference materi-

als. Moreover, this study attempts to conduct a

reasonable classification for yam landraces and pro-

vide scientific strategies for yam management.

Materials and methods

Plant material

Previous studies suggest that the production zone from

the North to the South of Yangtze River in China

represents a wide yam geographic distribution (Liu et al.

1993; Xu and Xu 1997). In the present study, a total of 21

yam landraces (61 individuals) from seven cultivated

populations in the wide zone, including all domesticated

species of Dioscorea, Dioscorea opposita Thunb.,

Dioscorea alata L., Dioscorea persimilis Prain et

Burkill, and Dioscorea fordii Prain et Burkill, were

collected for genetic analysis. Each landrace consisted

of two to five random individuals with approximately

5 g green and young leaves from locations at least

500 m apart. The botanical classification of samples was

based on morphological characters (Pei and Ding 1985)

and was determined by Jiangsu Institute of Botany,

Chinese Academy of Sciences. Detail information

including landraces, species, voucher numbers and

populations is listed in Table 1.

DNA extraction

DNA from about 50 mg silica gel dried leaves was

extracted using the improved cetyltrimethylammonium

Table 1 Yam landraces and species used in this study collected in the seven investigated populations

Landraces Sample code and size Species Voucher numbers Populations and abbreviations

Tiegunshanyao TIS 1–2 D. opposita Wu2010–Wx001 Wenxian County, Henan Province (WX)

Taigushanyao TGS 1–2 D. opposita Wu2010–Wx002

Huazishanyao HZS 1–2 D. opposita Wu2010–Wx003

Baiyushanyao BYS 1–2 D. opposita Wu2010–Wx004

Shuijinshanyao SJS 1–2 D. opposita Wu2010–Wx005

Qingyuanshanyao QYS 1–2 D. opposita Wu2010–Wx006

Hannanshanyao HNS 1–3 D. opposita Tao2011–NP001 Nanping City, Fujiang Province (NP)

Changdingshanyao CTS 1–2 D. opposita Tao2011–NP002

Nanpingshanyao NPS 1–2 D. opposita Tao2011–NP003

Lenanshanyao LNS 1–2 D. opposita Tao2011–NP004

Huaishanyao HSY 1–5 D. alata Li2010–Sx001 Shaxian County, Fujiang Province (SX)

No. 1 of shenshu SS1 1–3 D. alata Wu2011–Yq001 Yueqing City, Zhejiang Province (YQ)

No. 2 of shenshu SS2 1–3 D. alata Wu2011–Yq002







Guangdonghuaishan GDS 1–3 D. fordii Li2010–Sm002 Sanming City, Fujiang Province (SM)

GDS 4–6 D. fordii Wei2010–Gl001 Gaolou County, Zhejiang Province (GL)

Guangxihuaishan GXS 1–5 D. persimilis Wei2010–Ts002 Taoshan County, Zhejiang Province (TS)

Genet Resour Crop Evol (2014) 61:639–650 641

123

bromide (CTAB) method (Li et al. 2007). The DNA

quality was checked on a 1.0 % (w/v) agarose gel and

the concentration was measured by UV visible spectro-

photometer (Agilent 8453E, USA). All DNA samples

were diluted to 20 ng lL-1 and stored at -20 �C prior

to PCR amplification.

ISSR and SRAP amplification

Eleven ISSR primers (UBC primers set #9, University

of British Columbia, Canada) were chosen to for PCR

reaction based on band reproducibility (Table 2). ISSR

reactions were performed in a 20 lL final volume

containing 2.0 lL of 109 buffer (with Mg2?), 1.6 lL

of dNTP (2.5 mM), 0.2 lL of Taq Polymerase

(5 U lL-1), 1 lL of Primer (10 mM), and 3 lL of

DNA template (20 ng lL-1). Amplification was made

on PTC-200 TM programmable Thermal Controller

(Bio-Rad, USA), starting with 3 min at 94 �C, and then

35 cycles of 30 s at 94 �C, 45 s annealing at 53 �C and

90 s extension at 72 �C, with final extension at 72 �C

for 7 min. Further, a total of sixty SRAP primers (Li

and Quiros 2001) were used to test DNA amplification,

of which twenty primer combinations showed high

ability to detect polymorphism (Table 2). The SRAP

amplification mixture with a total volume of 20 lL

consisted of 2 lL of 109 buffer (with Mg2?), 1.6 lL

of dNTP (2.5 mM), 0.2 lL of Taq polymerase

(5 U lL-1), 1 lL of the forward primer (Me)

(10 mM), 1 lL of the reverse primer (Em) (10 mM),

and 3 lL of DNA template (20 ng lL-1). The ampli-

fication was programmed for an initial pre-denatur-

ation step of 5 min at 94 �C, followed by 5 cycles of

30 s denaturation at 94 �C, and then 30 s annealing

was carried out at 36 �C and 1 min extension at 72 �C,

the next procedure included 35 cycles of 30 s at 94 �C,

30 s annealing at 50 �C, and 1 min extension at 72 �C,

ending with a final extension of 5 min at 72 �C.

All PCR products were separated on 2.0 % (w/v)

agarose gels in 19 TBE buffer solution at 150 v voltage

for 30 min, and then stained using ethidium bromide

(EB) [Sangon Biotech (Shanghai) Co., China)]. The

separated DNA bands were visualized using the Alphal-

mager TM 2200 controller (Alpha, USA) and estimated

by comparing with 2,000 bp ladder molecular size

standard (Takara). To reduce deviation, the PCR ampli-

fications were performed twice and only clear repetitive

DNA bands were utilized in ISSR and SRAP analysis.

Data analysis

Three binary data matrixes from ISSR, SRAP and

ISSR ? SRAP, were respectively generated in terms

of the presence (1) or absence (0) with clear and

polymorphic bands. The ability of informative primers

to discriminate yam landraces was estimated by

calculating resolving power (Rp) (Prevost and Wil-

kinson 1999) according to the formula of Gilbert et al.

(1999), Rp ¼P

Ib, and Ib = 1 - (2 9 |0.5 - P|),

where p was the proportion of 21 landraces possessing

1 band. Assuming Hardy–Weinberg equilibrium, the

POPGENE software (version 1.31) was used to

calculate parameters such as the observed number of

alleles (Na), effective number of alleles (Ne), Nei’s

gene diversity (h), Shannon information index (I), the

percentage of polymorphic bands (PPB), the coeffi-

cient of genetic differentiation (Gst) and gene flow

(Nm), and there is a formula of Nm = 0.5 (1 - Gst)/

Gst (Slatkin and Barton, 1989). In light of the genetic

distances calculated from Euclidean similarity index,

the analysis of molecular variance (AMOVA) was also

conducted to assess variance component via the

ISSR ? SRAP data by using the WINAMOVA (ver-

sion 1.55) program (Excoffier 1993). Additionally,

unweighted pair group method arithmetic averages

(UPGMA) was adopted to cluster landraces into

original species based on the Dice similarity coeffi-

cients. The principal component analysis was also

performed based on the Jaccard’s similarity coeffi-

cients with help of the computer program, NTSYS-

pcVersion 2.1 (Rohlf 2000).

Results

Polymorphism and primer evaluation

The eleven selected ISSR primers produced a total of

150 bands with size ranging from 100 to 2,000 bp. Out

of these, 143 (95.3 %) bands were polymorphic with

an average of 13 polymorphic bands per ISSR primer

(Table 2). The number of polymorphic bands ranged

from 9 for UBC808 primer to 20 for UBC867 primer.

Further, the twenty SRAP primer combinations gen-

erated a total of 309 bands, of which 289 (93.5 %)

bands were polymorphic. The number of polymorphic

bands with SRAP primers ranged from 8 for Me4–

Em7 to 23 for Me5–Em7 with an average of 14.5


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(Table 2). The resolving power (Rp) of the eleven

ISSR primers ranged from 3.80 for primer UBC808 to

7.90 for primer UBC867 with a mean of 6.20. Three

ISSR primers (UBC841, UBC875, and UBC867)

possess the highest Rp values (7.21, 7.21, and 7.90,

respectively) and are each able to distinguish all 21

yam landraces. Interestingly, higher Rp was observed

for SRAP primer combinations, ranging from 3.28 for

Me5–Em4 to 11.84 for Me5–Em7 with a mean Rp of

6.77. Out of the twenty SRAP primer combinations,

three combinations (Me6–Em2, Me2–Em8, and Me5–

Em7) possess the highest Rp values (8.56, 10.23, and

11.84, respectively) and also have the ability to

distinguish all 21 yam landraces.

Genetic diversity

The genetic diversity parameters at both, individual

population level and the total population level (mean-

ing all investigated yam landraces) are shown in

Table 3. Based on the ISSR marker estimates, the

value of Nei’s gene diversity (H) and the Shannon

information index (I) were 0.29, and 0.45, respectively;

the percentage of polymorphic bands (PPB) was up to

95.3 % at total population level. Within the popula-

tions, the same change trend ranging from the lowest

(SM) to the highest (WX) was displayed for the value

of PPB, H and I. PPB ranged from 0 % to 51.3 %,

H ranged from 0 to 0.17, and I ranged from 0 to 0.27.

Table 2 List of ISSR primers and SRAP primer combinations

used to evaluate 21 yam landraces in China, including primer

sequence, number of recorded bands (Rb), number of poly-

morphic bands (Pb), the percent of polymorphism, number of

different landraces identified (Ni), and the resolving power of

primers (Rp)

Primer Rb Polymorphism Ni Rp

Pb %

ISSR 50 ? 30

UBC835 (AG)8YC 16 15 93.8 15 5.64

UBC839 (TA)8RG 13 12 92.3 16 6.13

UBC841 (GA)8YC 14 13 92.9 21 7.21

UBC845 (CT)8RG 13 13 100.0 20 6.66

UBC853 (TC)8RT 11 11 100.0 19 5.25

UBC857 (AC)8YG 12 11 91.7 16 5.25

UBC867 (GGC)5 20 20 100.0 21 7.90

UBC875 (CTAG)4 14 14 100.0 21 7.21

UBC808 (AG)8C 10 9 90.0 11 3.80

UBC809 (AG)8G 14 12 85.7 17 6.33

UBC815 (CT)8C 13 13 100.0 18 6.82

Total 150 143 195 68.20

Mean 13.6 13.0 95.3 18 6.20

SRAP

Me1 (50-BATA-30)–Em7

(30-DTGC-50)14 14 100.0 15 6.62

Me2 (50-BAGC-30)–Em7

(30-DTGC-50)12 12 100.0 10 5.67

Me2 (50-BAGC-30)–Em8

(30-DTGA-50)19 19 100.0 21 10.23

Me3 (50-BAAT-30)–Em2

(30-DCTG-50)15 14 93.3 16 7.77


(30-DCGA-50)15 11 73.3 13 5.97


(30-DCCA-50)17 13 76.5 13 5.93


(30-DAAC-50)15 14 93.3 15 5.80


(30-DTGC-50)9 9 100.0 12 4.07

Me4 (50-BACC-30)–Em7

(30-DCAA-50)9 8 88.9 9 3.67


(30-DCTG-50)17 16 94.1 19 7.25


(30-DCGA-50)16 15 93.8 18 7.67

Me5 (50-BAAG-30)–Em2

(30-DCTG-50)14 14 100.0 18 7.21


(30-DCCA-50)14 11 78.6 7 3.28


(30-DAAT-50)16 15 93.8 15 6.10


(30-DAAC-50)18 17 94.4 19 7.84

Table 2 continued

Primer Rb Polymorphism Ni Rp

Pb %


(30-DCAA-50)24 23 95.8 21 11.84

Me6 (50-BTGT-30)–Em1

(30-DCAA-50)17 17 100.0 16 6.52


(30-DCTG-50)20 19 95.0 21 8.56


(30-DGCA-50)14 14 100.0 20 7.64

Me5 (50-BAAG-30)–Em

10(30-DGCA-50)14 14 100.0 12 5.77

Total 309 289 310 135.41

Mean 15.5 14.5 93.5 16 6.77

R = (A, G); Y = (C, T); B = TGAGTCCAAACCGG;

D = GACTGCGTACGAATT; Me = the forward primer,

Em = the reverse primer


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The genetic diversity based on SRAP estimates was

similar to that observed for ISSR markers. Therefore,

in order to explain the genetic diversity and gene

differentiation precisely, the subsequent analyses were

carried out with ISSR ? SRAP data. The combination

of two marker types resulted in PPB of 94.1 % in all the

21 yam landraces investigated. The minimum genetic

distance of 0.0569 (not shown) was observed between

SM and GL populations because the same species, D.

fordii, is cultivated there. In contrast, the maximum

genetic distance of 0.4967 was found between WX and

GL as two different species, D. opposita and D. fordii,

are cultivated in these areas. The high level of

polymorphism and large range of genetic distance

from 0.0569 to 0.4967 indicates the existence of rich

genetic diversity among investigated yam populations.

The total genetic diversity among populations

(Ht = 0.3022 ± 0.0268) was significantly higher than

that within the populations (Hs = 0.0537 ± 0.0027).

The value of gene differentiation among populations

was 0.8222, which corresponded to the value of

0.1081 for gene flow. This pattern of variation

distribution was in agreement with the results obtained

by AMOVA analysis. In particular, 35.78 % of total

variation resided among populations with high signif-

icance (p \ 0.001*), and 23.83 % of this was varia-

tion within populations (Table 4). When all yam

individuals investigated were grouped according to

corresponding species, 40.39 % of total variation was

detected among the four yam species. Further, the

genetic diversity within D. opposita was the highest

with 62.3 % polymorphism (not shown). This was

followed by D. alata with 31.2 % polymorphism and

D. fordii was the lowest with only 5.9 % polymor-

phism. This result suggested that D. opposita and D.

alata were more prone to induced genetic differenti-

ation than the other two species.

Cluster analysis

UPGMA clustering of the 21 landraces based on the

Dice similarity is shown in Fig. 1a–c. Out of the three

kinds of UPGMA clustering, the analysis of ISSR

(Fig. 1a) reflected the geographic correspondence of

yam landraces. Landraces from the North of Yangtze

River, e.g., TIS, TGS, HZS, belonging to D. opposita,

formed one cluster (I). Whilst the landraces from the

South of Yangtze River grouped into another cluster

(II); they were further separated into four subgroups

representing the four yam species. It was noted that

some landraces belonging to the same species (D.

opposita) did not cluster together due to different

cultivation zones (WX, NP). The results of SRAP

markers (Fig. 1b) also supported that all landraces

were divided into two major groups (I, II). However,

unlike with ISSR analysis, the landraces of D.

opposita grouped together and comprised cluster I

with two subgroups containing the WX and NP

populations. The cluster II was composed of three

species of D. alata, D. fordii and D. persimilis,

forming three subgroups. In comparison the combi-

nation of ISSR ? SRAP markers (Fig. 1c), had

Table 3 Genetic diversity of 21 yam landraces in China within

and among seven investigated populations, including observed

number of alleles (Na), effective number of alleles (Ne), Nei’s

gene diversity (H), Shannon information index (I), and the

percentage of polymorphic band (PPB)

POP ISSR SRAP ISSR ? SRAP

Na Ne H I PPB (%) Na Ne H I PPB (%) Na Ne H I PPB (%)

WX 1.51 1.30 0.17 0.27 51.3 1.36 1.23 0.13 0.20 35.9 1.41 1.25 0.15 0.22 41.0

SX 1.10 1.06 0.04 0.05 10.0 1.00 1.00 0 0 0 1.03 1.02 0.01 0.02 3.3

SM 1.00 1.00 0 0 0 1.00 1.00 0.01 0.01 0.3 1.00 1.00 0 0 0.2

NP 1.35 1.21 0.13 0.19 35.3 1.16 1.11 0.06 0.09 15.5 1.22 1.14 0.08 0.12 22.0

YQ 1.28 1.19 0.11 0.16 28.0 1.22 1.13 0.08 0.11 21.7 1.24 1.15 0.09 0.13 23.8

TS 1.23 1.17 0.10 0.14 23.3 1.07 1.05 0.03 0.04 6.8 1.12 1.09 0.05 0.07 12.2

GL 1.01 1.01 0.01 0.01 0.7 1.00 1.00 0 0 0 1.00 1.00 0 0 0.2

Total level 1.95 1.48 0.29 0.45 95.3 1.94 1.47 0.28 0.44 93.5 1.94 1.47 0.29 0.44 94.1

The total level means diversity of all investigated yam landraces; the mean value in this table


123

similar classification pattern to that of SRAP markers.

The comprehensive dendrogram based on ISSR ? S-

RAP markers revealed more distinct genetic relation-

ships among landraces and all landraces could be

accurately classified into the four yam species. This

classification was also well illustrated by the principal

component analysis for the 61 individuals using

Jaccard’s similarity coefficients. Plotting of the first

and second components clearly separated the yam

landraces into four groups (Fig. 2). Moreover, both

group A (D. opposita) and group B (D. alata) were

further divided into two subgroups (I, II) according to

two planting zones. Genetic distances among the four

yam species were measured when individuals were

grouped in terms of their corresponding species. D.

opposita and D. alata were closely related with the

minimum genetic distance of 0.2479 (not shown),

while D. fordii and D. persimilis were most divergent

species with the maximum distance of 0.4255 (not

shown).

Discussion

Geographic distribution and clustering

The number of yam species domesticated in China has

always been controversial (Huang 2005). A previous

study suggested that only two yam species, D.

opposita and D. alata, were domesticated for food

production (Cai et al. 1999). However, our research

showed that four domesticated species including D.

opposita, D. alata, D. persimilis, and D. fordii, are

widely used as both, food and medicine (Wu et al.

2009). Of these four species, D. opposita was deemed

to be the most prevalent species and thought to be only

cultivated in the North of Yangtze River (Xu and Xu

1997). However, our investigation revealed that some

representative yam landraces of D. opposita (e.g.,

HNS, NPS, LNS) have recently appeared through

domestication by local farmers in southern region and

they comprised a single subgroup (Fig. 1). This result

may indicate that the southern region has formed a

new independent cultivation zone of D. opposita due

to positive breeding programs. Our UPGMA cluster-

ing supported the fact that landraces collected from the

same locality always share a closer genetic relation-

ship and form an independent subgroup (Fig. 1),

probably in terms of these landraces domesticated in

their production zone are progeny of the same original

parents. Moreover, this relationship of clustering was

highly consonant with the partition rule of geographic

distance (Loveless and Hamrick 1984).

Identification based on ISSR and SRAP markers

Species identification of yams has always been a

subject of speculation in China. Some landraces may

bear the same name but actually they belong to

different species. For instance, the so-called ‘‘huan-

shanyao’’ as medical resource is made up of two

species involving D. opposita and D. alata (Xiao

2002). However, only the use of ‘‘Dioscorea rhizome’’

from D. opposita is permitted as medicinal species and

listed in Chinese pharmacopoeias (Pharmacopoeia of

China 2010). In yam, cultivar identification via

molecular marker-based analyses will be very useful

because genetic variation is fixed within a line. Some

researchers have accurately classified the traditional

cultivars of Jamaican yams and Guinea yams using

RAPD and AFLP markers, respectively (Asemota et al.

1996; Dansi et al. 2000a). Previously, Prevost and

Wilkinson (1999) concluded that the nature of the

relationship between Rp and genotype diagnosis is a

seemingly linear correlation (r2 = 0.65). In compari-

son, the ISSR and SRAP primers used in this study

demonstrated a strong ability to identify yam landraces

in China with high Rp values (Table 2). We found

three ISSR primers (UBC841, UBC875 and UBC867)

and three SRAP primer combinations (Me6–Em2,

Me2–Em8, and Me5–Em7), with the Rp over 7.21, and

8.56, respectively, that are each able to differentiate all

Table 4 Analysis of molecular variance (AMOVA) for seven

yam investigated populations in China, considering four spe-

cies according to the difference of species in cultivated popu-

lations, species I (D. opposita from WX and NP), species II (D.

alata from SX and YQ), species III (D. fordii from SM and

GL), and species IV (D. persimilis from TS), including degrees

of freedom (df), variance-components estimates (VC), per-

centage of the total variance contributed by each component

(% total), and P value (1,000 permutations)

Source of variation df VC % Total P value

Among species 3 74.869 40.39 \0.001*

Among populations

within species

3 66.316 35.78 \0.001*

Within populations 54 44.178 23.83

Total 60 185.363 100.00


123


123

yam landraces. Also, we demonstrated a greater linear

correlation (r2 = 0.75 for ISSR primers, and r2 = 0.79

for SRAP primer combinations) between the Rp and

the number of yam landraces identified than that

reported by Prevost and Wilkinson (1999).

The strong relationship meant it was possible to

estimate how many landraces could be identified by

calculating the Rp of a hypothetical primer. All the 21

yam landraces from China could be distinguished and

divided into four yam species only using the primers

with a given high Rp values (Figs. 1, 2). The

classification of four yam species, D. opposita, D.

alata, D. persimilis, and D. fordii, was in agreement

with morphological taxonomy of Ding and Gilbert

(2000). These primers with high Rp values may

therefore be routinely used for yam landrace identi-

fication and serve as an instrument to stop misidenti-

fication of medicinal yams.

Genetic diversity and gene flow

The knowledge about the genetic diversity of crops is

essential in genetic improvement programs. In this

study, in the light of high quality marker profiles

obtained (Additional file), the level of polymorphism

of 95.3 and 93.5 % was respectively detected by ISSR

and SRAP markers. This revealed a relatively high

level of genetic diversity among yam landraces in

China. The extent of landrace diversity detected in this

study is comparable to an earlier report involving

Chinese yams (Zhou et al. 2005; Hua et al. 2009), and

it is higher than previous reports by both Zhou et al.

(2005) (81.0 %) and Hua et al. (2009) (88.5 %). The

explanation for diverse yam landraces maintained by

farmers may be attributed to environmental adaptation

and the length of growing period. This finding further

highlights the importance of traditional farmers in

preserving genetic diversity among the landraces.

Also, an understanding of population genetic

structure is essential for genetic introgression and to

design appropriate conservation strategies (Ngu et al.

2010). In this study, partitioning of genetic diversity

by AMOVA revealed a greater variation among

Fig. 2 Plot of sixty-one

individuals of 21 yam

landraces in China

according to the two first of

the principal component

analysis (PCA) based on the

combination of ISSR and

SRAP markers, revealing

four distinct groups, A (D.

opposita), B (D. alata),

C (D. persimilis), and D (D.

fordii). Each individual

described by a

corresponding code is given

in Table 1

Fig. 1 UPGMA dendrogram of 21 yam landraces in China

based on ISSR (a), SRAP (b) and the combination of

ISSR ? SRAP (c), and dendrogram revealing four yam species,

D. opposita, D. alata, D. fordii and D. persimilis. Each landrace

described by a corresponding code is given in Table 1

b


123

populations (35.78 %) than within populations

(23.83 %). However, in contrast a previous study by

Tamiru et al. (2007), in the study of yam germplasm

from Ethiopia and their related cultivated Dioscorea

species, found most of the genetic variation (81 %)

existed within rather than among populations. This

may reflect a greater spatial separation of Dioscorea

species in China. Meanwhile, owing to our sampling

technology, four different yam species were collected

from seven investigated populations. Therefore, on the

other hand, the high variation among populations was

also caused by genetic difference among the four yam

species, 40.39 % of total variation was observed

among them. Further, the lower variation observed

within population implied low diversity level for

individual yam species (Table 3). Of the four yam

species, only D. opposita and D. alata possessed

moderate genetic diversity. In practice, the two species

with rich phenotypic traits were commonly used as

parent materials for directed breeding, e.g., such

landraces as TIS, TGS and HZS have been success-

fully selected in recent years (Huang 2005). Compar-

atively, D. persimilis and D. fordii have relatively low

genetic diversity and are not suitable as breeding

materials. This result is consistent with our previous

study (Wu et al. 2009).

The lack of genetic diversity observed for individual

yam species is likely a substantial consequence of two

cultural practices. Primarily, it is thought that yam is

deemed as a vegetative propagation crop and regener-

ation occurs mostly in sprouting from tubers and bulbs

(Gucker and Corey 2009). Thus, individuals are

usually highly homozygous through repeated vegeta-

tive propagation cycles, maintaining less genetic

variability at the individual level (Pelsy et al. 2010).

The emergence of variant plants arising as a result of

genetic recombination and seed dispersal, however, is

unlikely, since the occurrence of flowering and fruiting

in these cultivated species was not observed during

their whole growth (D. persimilis occasionally pro-

duced only a few flowers according to our investiga-

tion). This isolation of sexual organ may be responsible

for limits of genetic transfer within species and similar

results were also obtained in other crops (Valbuena-

Carabana et al. 2008; De Carvalho et al. 2011).

Alternatively, yam species have been subjected to a

strong selection through obtaining some modifications

in tuber form, color, size and taste, which is likely to

result in loss of some special gene information in the

process of their asexual propagation cycles. The

breeding program served as a double-edged sword at

the beginning of domestication. Although it provided a

benefit by ensuring true breeding cultivars, it also

threatened yam genetic diversity by introduction of

improved varieties. For example a study of Guinea

yams (Mengesha et al. 2013), reported that most of the

allelic diversity was found within wild relatives rather

than cultivar species. Wild yams, therefore, are

important for yam breeding by acting as desirable

parents of useful genes. The large scale production of

potentially best genotypes has been acquired by

hybridizing wild and cultivated species via sexual

reproduction (artificial pollination) (Scarcelli et al.

2006). Also, high level of resistance to anthracnose

from the first generation (F1) in white yam (D. alata)

was introduced into new varieties by genetic transfor-

mation (Mignouna et al. 2002).

Distribution of genetic variability and the extent of

gene flow between populations are important for

understanding genetic evolution. In this study, we

detected a large genetic difference among seven

investigated populations, the Gst of 0.8222 lead to

Nm = 0.1081, suggesting a low gene flow among

populations. This may be explained by two directly

related facts. Firstly, the degree of differentiation

among populations is affected by the rate at which

genes are carried between populations by the migration

of pollen or seeds. In general, plant species with higher

frequency of seed and pollen movement result in less

genetic differentiation than species with restricted

gene flow (Ellstrand 1992; Hamrick and Nason 1996).

The gene flow in this study is hindered by flowers and

seed isolation between several cultivated species.

Secondly, there are four different yam species domes-

ticated and widely distributed in China, namely, D.

opposita, D. alata, D. fordii and D. persimilis. It is

known that different species under natural conditions

are usually separated by more or less strong barriers to

gene flow. Additionally, we also did not find any

heterozygous varieties through outcrossing between

different species during cultivation, or introduced from

different genotypes through genetic transformation. In

fact, yam breeding programs in China have mainly

focused on direct selection within species. Thus, the

possibility of gene flow between yam species from

China is relatively slim.


123

Implications for conservation and improvement

The results of the present study demonstrate that ISSR

and SRAP molecular markers are an attractive

approach to identify yam landraces and to assess their

genetic relationships. The relatively high level of

genetic diversity displayed by yam landraces was

responsible for large genetic variation of different yam

species cultivated in different environment. This study

also revealed distinctly low level of diversity within

individual yam species (e.g., D. opposita, D. alata, D.

persimilis, D. fordii) due to the favorable vegetative

propagation method and intense cultivar breeding

programs. Therefore, it is crucial to enhance genetic

diversity in cultivated yams through developing long-

term strategies, such as modifying breeding programs

through increasing utilization of wild species, con-

serving core germplasm, and establishing gene

resource pools. Our observation about similarity in

yams from same geographic locations may provide

some insight for dissecting the potential relationship

between genes of yam landraces and environmental

conditions.

Acknowledgments The present study was supported by the

Agricultural Science and Technology Program of Wenzhou in

China (Grant No. N20100003), Zhejiang Provincial Key

Laboratory for Genetic Improvement and Quality Control of

Medicinal Plants (Grant No. 2011E10015). We are grateful to

RuiAn Agriculture and Foresty Bureau (Ruian city, Zhejiang

Province), Sanming Agricultural Science Institute (Sanming

city, Fujian Province), Wenxian Agricultural Science Institute

(Wenxian City, Henan Province), for positive help with

sampling yam landraces. Many thanks to Pro. Xin-Chun Lin

and Xiao-Xia Li, for their help with PCR experiments.

References

Asemota HN, Ramer J, Lopez-Peralta C, Weising K, Kahl G

(1996) Genetic variation and cultivar identification of

Jamaican yam germplasm by random amplified polymor-

phic DNA analysis. Euphytica 92:341–351

Bahieldin A, Ahmed IA, Gad El-Karim GA, Eissa HF, Mahfouz

HT, Saleh OM (2006) DGGE-RAPD analysis as a useful

tool for cultivar identification. Afr J Biotechnol 5:566–569

Board of pharmacopoeia of PR China (2010) Pharmacopoeia of

the People’s Republic of China, part I. Board of pharma-

copoeia of PR China, Beijing, p 27

Budak H, Shearman RC, Parmaksiz I, Dweikat I (2004) Com-

parative analysis of seeded and vegetative biotype buffa-

lograsses based on phylogentic relationship using ISSRs,

SSRs, RAPDs, and SRAPs. Theor Appl Genet 109:280–288

Burkill IH (1960) The organography and evolution of Dio-

scoreaceae, the family of yams. J Linn Soc Lond Bot

56:319–412

Cai JH, Yan JZ, Huang XH, Ye SJ, Jiang LW, Gu ZZ (1999) A

taxonomical study on varietal resource of Dioscorea spe-

cies. Acta Jiangxi Agric Univ 21:53–57

Coursey DG (1967) Yams: an account of the nature, origins,

cultivation and utilization of the useful members of the

Dioscoreaceae. Longmans, Green, London

Dansi A, Mignouna HD, Zoundjihekpon J, Sangare A, Ahous-

sou N, Asiedu R (2000a) Identification of some Benin

Republic’s Guinea yams (Dioscorea cayenenis/Dioscorea

rotundata complex) cultivars using randomly amplified

polymorphic DNA. Genet Resour Crop Evol 47:619–625

Dansi A, Mignouna HD, Zoundjihekpon J, Sangare A, Asiedu R,

Ahoussou N (2000b) Using isozyme polymorphism to

assess genetic variation within cultivated yams (Dioscorea

cayenenis/Dioscorea rotundata complex) of the Republic

of Benin. Genet Resour Crop Evol 47:371–383

De Carvalho VM, Marochio CA, Mangolin CA, Da Silva

Machado MDFP (2011) The use of esterase polymorphism

for analysis of the genetic diversity and structure of stevia

(Stevia rebaudiana (Bert.) Bertoni) populations. Biochem

Syst Ecol 39:594–599

Ding Z, Gilbert MG (2000) Flora of China, vol 24 (Dioscorea-

cea). In: Wu Z, Raven PH (eds) Flagellariaceae through

Marantaceae. Missouri Botanical Garden, St. Louis,

pp 276–296

Dumont R, Vernier P (2000) Domestication of yams (Dioscorea

cayenensis-rotundata) within the Bariba ethnic group in

Benin. Outlook Agric 6:137–142

Ellstrand N (1992) Gene flow by pollen: implications for plant

conversation genetics. Oikos 63:77–86

Excoffier L (1993) Analysis of molecular variance. Version 1.5.

Genetics and Biometry Laboratory, University of Geneva,

Geneva

Gilbert JE, Lewis RV, Wilkinson MJ, Galigari PDS (1999)

Developing and appropriate strategy to assess genetic

variability in plant germplasm collections. Theor Appl

Genet 98:1125–1131

Gucker CL (2009) Dioscorea spp. In: Fire Effects Information

System, [Online]. Department of Agriculture, Forest Ser-

vice, Rocky Mountain Research Station, Fire Sciences

Laboratory (Producer). http://www.fs.fed.us/database/feis/.

Accessed 9 Dec 2011

Hahn SK (1995) Yams: Dioscorea spp. (Dioscoreaceae). In:

Smartt J, Simmonds NW (eds) Evolution of crop plants.

B. Longman, Essex, pp 112–120

Hamrick JL, Nason JD (1996) Consequence of dispersal in

plants. In: Rhodes OE, Ronald KC, Smith MH (eds) Pop-

ulation dynamics in ecological space and time. Chicago,

pp 203–235

Hua SM, Tu QC, Lei FG (2009) Genetic diversity of Dioscorea

polystachya Turcz. revealed by RAPD markers. J Plant

Resour Environ 10:1995–2000

Huang WH (2005) Non-pollution and standardization cultiva-

tion for yam. Beijing, pp 16–44

Li G, Quiros CF (2001) Sequence-related amplified polymor-

phism (SRAP) a new marker system based on a simple

PCR reaction: its application to mapping and gene tagging

in Brassica. Theor Appl Gent 103:455–461


123

http://www.fs.fed.us/database/feis/

Li MJ, Xu X, Zhang XL, Liu YK (2007) Isolation of genomic

DNA and optimization of RAPD conditions of Dioscorea

opposita Thunb. J Henan Norm Univ 35:140–143

Liu P, Guo SL, Lu HF, Xie XW, Wu XY (1993) A summary on

of the study on Chinese Dioscorea spp. J Zhejiang Norm

Univ 16:100–106

Loveless MD, Hamrick JL (1984) Ecological determinants of

genetic structure in plant populations. Ann Rev Ecol Syst

15:65–95

Mengesha WA, Demissew S, Fay MF, Smith RJ, Nordal I,

Wilkin P (2013) Genetic diversity and population structure

of Guinea yams and their wild relatives in South and South

West Ethiopia as revealed by microsatellite markers. Genet

Resour Crop Evol 60:529–541

Mignouna HD, Dansi A (2003) Yam (Dioscorea spp.) domes-

tication by the Nago and Fon ethnic groups in Benin. Genet


Mignouna HD, Mank RA, Ellis THN, Van Den Bosch N, Asiedu

R, Abang MM, Peleman J (2002) A genetic linkage map of

water yam (Dioscorea alata L.) based on AFLP markers

and QTL analysis for anthracnose resistance. Theor Appl

Genet 105:726–735

Nascimento WF, Rodrigues JF, Koehler S, Gepts P, Veasey EA

(2013) Spatially structured genetic diversity of the Amer-

indian yam (Dioscorea trifida L.) assessed by SSR and

ISSR markers in Southern Brazil. Genet Resour Crop Evol.

60:2405–2420

Ngu MS, Sabu KK, Lim LS, Abdullah MZ, Wickneswari R

(2010) Genetic structure of Oryza rufipogon Griff. natural

populations in Malaysia: implications for conservation and

genetic introgression of cultivated Rice. Trop Plant Biol

3:227–239

Okoli OO (1991) Yam germplasm diversity, use and prospects

for crop improvement in Africa. In: Crop genetic resources

of Africa, vol 2. IITA/IBPGR/UNER/CNR, Ibadan, Nige-

ria, pp 109–117

Pei J, Ding ZZ (1985) Flora of China, vol. 16 (Dioscoreacea).

Beijing, pp 103–119

Pelsy F, Hocquigny S, Moncada X, Barbeau G, Forget D,

Hinrichsen P, Merdinoglu D (2010) An extensive study of

the genetic diversity within seven French wine grape

variety collections. Theor Appl Genet 120:1219–1231

Prevost A, Wilkinson MJ (1999) A new system of comparing

PCR primers applied to ISSR fingerprinting of potato

cultivars. Theor Appl Genet 98:107–112

Qin HZ, Zhang MZ, Ling PP, Ding ZZ, Dou FP (1985) A

cytotaxonomic study on Chinese Dioscorea L.—the chro-

mosome numbers and their relation to the origin and evo-

lution of the genus. Acta Phytotax Sin 23:11–18

Ramser J, Weising K, Lopez-peralta C, Terhalle W, Terauchi R,

Kahl G (1997) Molecular marker based taxonomy and

phylogeny of Guinea yam (Dioscorea rotundata–D. cay-

enensis). Genome 40:903–915

Rohlf FJ (2000) NTSYSpc: numerical taxonomy and multi-

variate analysis system. Exeter Software, New York

Rubatzky VE, Yamaguchi M (1997) World vegetables: princi-

ples, production, and nutritive values (2nd edn). New York,

pp 162–182

Sartie A, Asiedu R, Franco J (2012) Genetic and phenotypic

diversity in a germplasm working collection of cultivated

tropical yams (Dioscorea spp.). Genet Resour Crop Evol

59:1753–1765

Scarcelli N, Tostain S, Mariac C, Agbangla C, Da O, Berthaud J,

Pham JL (2006) Genetic nature of yams (Dioscorea spp.)

domesticated by farmers in Benin (West Africa). Genet


Shao QS, Guo QS, Deng YM, Guo HP (2010) A comparative

analysis of genetic diversity in medicinal Chrysanthemum

morifolium based on morphology, ISSR and SRAP mark-

ers. Biochem Syst Ecol 38:1160–1169

Shu P (1987) Study on pollen morphology from Dioscorea

genus in China. Acta Phytotax Sin 25:357–365

Slatkin M, Barton NH (1989) A comparison of three indirect

methods for estimating average levels of gene flow. Evo-

lution 36:1349–1368

Tamiru M, Becker HC, Maass BL (2007) Genetic diversity in

yam germplasm from Ethiopia and their relatedness to the

main cultivated Dioscorea species assessed by AFLP

markers. Crop Sci 47:144–153

Tostain S, Agbangla C, Scarcelli N, Mariac C, Daınou O,

Berthaud J, Pham JL (2007) Genetic diversity analysis of

yam cultivars (Dioscorea rotundata Poir.) in Benin using

simple sequence repeat (SSR) markers. Plant Genet Resour

5:71–81

Valbuena-Carabana M, Gonzalez-Martinez SC, Gil L (2008) A

case study in Quercus pyrenaica Willd. from central Spain.

For Ecol Manag 254:225–232

Wilkin P, Schols P, Chase M, Chayamarit K, Furness CA, Hu-

ysmans S, Rakotonasolo F, Smets E, Thapyai C (2005) A

plastid gene phylogeny of the yam genus, Dioscorea: roots,

fruits and Madagascar. Syst Bot 30:736–749

Wu ZG, Leng CH, Tao ZM, Wei YH, Jiang CX (2009) Genetic

diversity of Dioscorea alata L. based on ISSR analysis.

J Chin Mater Med 34:3017–3019

Xia CX, Gao SL, Zhang CY, Huang XS (2004) Analysis of the

chemical components and isomorphic amylase among

different local cultivars of Diocorea opposita. J Plant Re-

sour Environ 13:21–24

Xiao PG (2002) Modern Chinese Materia Medica, vol I. Beijing,

p 740

Xu GJ, Xu LS (1997) Species systematization and quality

evaluation of commonly used Chinese traditional drugs,

South-China, vol II. Fuzhou, pp 449–471

Zhou YQ, Jing JZ, Li ZY, Hao J, Jia JF, Zhang BH, Hao JG

(2005) Genetic diversity of yam (Dioscorea opposita

Thunb.) detected by ISSR markers. Acta Bioe Exp Sin

38:324–329


123

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Genetic diversity analysis of yams (Dioscorea spp.) cultivated in China using ISSR and SRAP markers