Barley Genetics Newsletter (2005) 35:9-22

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Genetic diversity in barley from west China

based on RAPD and ISSR analysis

Yong-Cui Hou, Ze-Hong Yan, Yu-Ming Wei, You-Liang Zheng\*

Triticeae Research Institute, Sichuan Agricultural University,
Dujiangyan, 611830, China

\*Corresponding author, E-mail: ylzheng@sicau.edu.cn

Abstract

Two types of molecular markers, random amplified polymorphic DNA (RAPD)
and inter-simple sequence repeat (ISSR), were assayed to determine the
genetic diversity of 46 barley accessions, including 27 landraces of *H.
vulgare* ssp *vulgare* (HV), 6 accessions of *H. vulgare* ssp.
*spontaneum* (HS) and 13 accessions of *H. vulgare* ssp. *agriocrithon*
(HA), from west China. A high level of polymorphism was found with both
RAPD and ISSR markers, and the mean polymorphism information content
(PIC) values were 0.574 and 0.631 for RAPD and ISSR markers,
respectively. In RAPD analyses, 84 out of 109 bands (77.06%) were
polymorphic. The number of alleles ranged from 2 to 8 per primer, with
an average of 4.19 per primer. The RAPD-based genetic similarity
(RAPD-GS) ranged from 0.753 to 0.980, with the mean of 0.864. In ISSR
analyses, a total of 107 alleles were detected, among which 105 alleles
(98.13%) were polymorphic. The number of alleles per primer ranged from
2 to 10 with an average of 5.94 alleles per ISSR primer. The
ISSR-derived genetic similarity (ISSR-GS) ranged from 0.212 to 0.931,
with the mean of 0.674. ISSR was better than RAPD to detect genetic
diversity among the barley accessions. A poor correlation (r = 0.113)
was found between both sets of genetic similarity data, suggesting that
both sets of markers revealed unrelated estimates of genetic
relationships. Cluster analyses indicated that all 46 barley accessions
could be distinguished by both RAPD and ISSR markers.

**Key words:** barley, RAPD markers, ISSR markers, genetic diversity,
west China

# Introduction

Barley, *Hordeum vulgare* L., is one of the principal cereal crops in
the world and is cultivated in all temperate areas (von Bothmer et al.
1995). Wild barleys, *H*. *vulgare* ssp. *spontaneum* and *H*. *vulgare*
ssp *agriocrithon*, are the primary gene pool of cultivated barley (*H.
vulgare* ssp. *vulgare*). The total number of barley accessions in the
Genbanks, including redundant materials, is estimated to be about
280,000 (Plucknett et al. 1987). China is known to be a genetic
diversity center of barley and rich in both landrace and wild relatives
of barley (Yang et al., 1987). The wild relatives of barley have been
considered to be a large reservoir of genetic diversity. It is known
that wild barley populations possess high genetic variation in several
useful characters, including earliness, biomass and yield, protein
content and a high proportion of resistant genotypes against powdery
mildew and leaf rust (Nevo 1992). Due to its compatibility and full
interfertility with cultivated barley, these wild species have been used
as a source of important genes for cultivar development via
interspecific crosses (Dávila et al. 1999a). Modern plant breeding and
agriculture system have narrowed the genetic base of cultivated barley
(Nevo et al. 1986). Therefore, the modern varieties are more genetically
homogeneous and more exposed to pathogens and adverse environmental
conditions (Asins and Carbonell 1989). This has promoted the search for
new sources of variation that might be of use in plant breeding
programs, and many national and international organizations have
stressed the need for the collection, conservation and use of
wild-species relatives of cultivated species and the endemic varieties
(Brown et al. 1990). Assessment of the extent of genetic variability
within barley, including the wild relatives, is fundamental for barley
breeding and the conservation of genetic resources, and is particularly
useful as a general guide in the choice of parents for breeding hybrids.

Molecular markers have been proved to be valuable tools in the
characterization and evaluation of genetic diversity within and between
species and populations. It has been shown that different markers might
reveal different classes of variation (Powell et al. 1996; Russell et
al. 1997). It is correlated with the genome fraction surveyed by each
kind of marker, their distribution throughout the genome and the extent
of the DNA target which is analyzed by each specific assay (Dávila et
al. 1999b). The advent of the polymerase chain reaction (PCR) favored
the development of different molecular techniques such as random
amplified of polymorphic DNA (RAPD), simple sequence repeats (SSR or
microsatellite), sequence tagged sites (STS), random amplified
microsatellite polymorphism (RAMP) and inter-simple sequence repeat
polymorphic DNA (ISSR), and so on (Saiki et al. 1988; Welsh and
McCleland 1990; Williams et al. 1990; Akkaya et al. 1992; Tragoonrung et
al. 1992; Zietkiewicz et al. 1994; Wu et al. 1994; Nagaoka and Ogihara
1997). These molecular markers had been used in barley for detecting
genetic diversity, genotype identification, genetic mapping (Sánchez de
la Hoz et al. 1996; Matus and Hayes 2002; Dávila et al. 1999a, 1999b;
Dávila et al. 1998; Tragoonrung et al. 1992; Tanyolac 2003; Fernández et
al. 2002; Struss and Plieske 1998). Of these techniques, RAPD has
several advantages, such as simplicity of use, low cost, and the use of
small amount of plant material, etc. RAPDs were proved to be useful as
genetic markers in the case of self-pollinating species with a
relatively low level of intraspecific polymorphism, such as hexaploid
wheat (Devos and Gale 1992; Joshi and Nguyen 1993) and cultivated barley
(Barua et al. 1993; Chalmers et al. 1993; Tinker et al. 1993). ISSR
markers, which involve PCR amplifications of DNA using a primer composed
of a microsatellite sequence anchored at 3' or 5' end by 2-4 arbitrary,
could be used to assess genetic diversity (Qian et al. 2001). ISSRs have
been used for cultivar identification in maize (Kantety et al. 1995;
Pejic et al 1998), potatoes (Prevost and Wilkinson 1999), trifoliate
orange (Fang et al. 1997), wheat (Nagaoka and Ogihara 1997), bean
(Métais et al. 2000), *Diplotaxis* (Martín and Sánchez-Yélamo 2000) and
barley (Fernández et al. 2002; Tanyolac 2003).

To date, knowledge regarding the amount of genetic variation and genetic
relationship at molecular marker in Chinese barley germplasm is
available using RAPD (Shi et al. 2004; Chen et al. 2000a, 2000b; Hong et
al. 2001) and SSR (Feng et al. 2003) markers. The objectives of this
study are to (1) reveal the ISSR-based genetic diversity in a barley
germplasm from western China, (2) compare RAPD and ISSR diversity in the
studied materials, and (3) assess the genetic diversity within the
selected accessions of the barley landraces as compared to that in its
wild relatives by using RAPD and ISSR molecular markers.

# 

# Materials and methods

Plant material and DNA extraction. A total of 46 barley accessions from
44 locations of west China were used in this study (Table 1). There were
27 landraces of *H. vulgare* ssp *vulgare* (HV), 6 wild relative forms
of *H. vulgare* ssp. *spontaneum* (HS) and 13 wild relative forms of *H.
vulgare* ssp. *agriocrithon* (HA). Genomic DNA was extracted from a bulk
sampling of a minimum of ten individuals for each accession according to
Sharp et al. (1988).

RAPD and ISSR analysis. A total of twenty-six 10-mer oligonucleotides
with arbitrary sequence from Operon (kits A, B, H and R) were used in
RAPD analysis (Table 2) and 18 primers based on dinucleotide,
tetranucleotide or pentanucleotide repeats were used in ISSR analysis
(Table 3). The PCR reaction mixture consisted of 20-50ng genomic DNA,
1×PCR buffer, 2.0 mmol/L MgCl~2~, 100 µmol/L of each dNTP, 0.1 µmol/L
primer and 1U *Taq* polymerase in a 25μL volume. The amplification
protocol was 94 ºC for 4 min to pre-denature, followed by 45 cycles of
94 ºC for 1 min, 36 ºC (for RAPD analysis) or 50 ºC (for ISSR analysis)
for 1 min and 72 ºC for 1 min, with a final extension at 72 ºC for 10
min. Amplification products were fractionated on 1% (for RAPD analysis)
or 2% (for ISSR analysis) agarose gel.

Data analysis. RAPD and ISSR data were scored for presence (1), absence
(0) or as a missing observation (9), and each band was regarded as a
locus. Two matrices, one for each marker, were generated. The genetic
similarities (GS) were calculated according to Nei and Li (1979): GS =
2N~ij~/(N~i~+N~j~), where N~ij~ is the number of bands present in both
genotypes i and j, N~i~ is the number of bands present in genotypes i,
and N~j~ is the number of bands present in genotype j. Based on the
similarity matrix, a dendrogram showing the genetic relationships
between genotypes, was constructed using the unweighted pairgroup method
with arithmetic average (UPGMA) (Sneath and Sokal, 1973) though the
software NTSYS-pc version1.80 (Rohlf, 1993). Polymorphic information
content (PIC) values were calculated for each RAPD and ISSR primer
according to the formula: PIC = 1-Σ(P~ij~)^2^, where P~ij~ is the
frequency of the i^th^ pattern revealed by the j^th^ primer summed
across all patterns revealed by the primers (Botstein et al. 1980). The
similarity between matrices based on different marker system (RAPD and
ISSR) were calculated using the standardized Mantel coefficient (Mantel
1967). The significance level for the correlation coefficient was
calculated following Sokal and Rohlf (1995).

# 

# Results

In RAPD analysis, a total of 109 bands were detected, among which 84
bands (77.06%) were polymorphic with the mean of 3.23 per primer (Table
2). For each primer, the number of bands ranged from 2 to 8, with an
average of 4.19. The average polymorphic information content (PIC) was
0.574, ranging from 0.156 to 0.894. The lowest and the highest PIC
values were recorded for primer OPH-13 and OPH-12, respectively.

In ISSR analysis, a total of 107 bands were observed, with 5.94 bands
per primer (Table 3). One hundred five out of 107 bands (98.13%) were
polymorphic, among which 2 to 10 polymorphic bands were detected by each
primer. The average PIC was 0.636, and the lowest and highest PIC values
were 0.351 (ISSR16) and 0.874 (ISSR18), respectively. Four ISSR primers
(i.e. ISSR18, ISSR7, ISSR6 and ISSR8) had the higher PIC values.

All the 109 bands, generated from 26 RAPD primers, were subjected to
calculate the genetic similarity index (RAPD-GS) among the 46 accessions
(Table 4.). The RAPD-GS value ranged from 0.735 to 0.980, with the mean
of 0.864. The highest genetic similarity was found between HV accession
B65 (from Xizang) and HV accession B21 (from Sichuan), while the lowest
genetic similarity was observed between HV accession B76 (from Sichuan)
and HA accession ZYM0531 (from Xizang). It was found that the genetic
similarities within the HV and HA groups were equal to that within HS
group.

The ISSR-derived data were subjected to calculate the genetic similarity
(ISSR-GS) (Table 4). The genetic similarity coefficient varied between
0.212 and 0.931, with the average of 0.674. The minimum GS value derived
between HV accession B22 (from Xizang) and HA accession ZYM0834 (from
Xizang), while the maximum GS value derived between HV accession B22
(from Xizang) and HV accession B40 (from Sichuan). The average genetic
similarity within HS accessions (0.645), HA accessions (0.623) and all
accessions (0.674) exhibited equivalent to each other, and the average
genetic similarity within HV accessions (0.716) was higher than that of
all accessions. In contrast to RAPD-GS, the ISSR-GS within HS accessions
(0.645) and HA accessions (0.623) were significant lower than that
within HV (0.716). It indicated that the wild barley groups were more
diverse than the landraces group. The average genetic similarity values
based on ISSR markers were lower than that of RAPD markers both within
landraces and wild barley accessions, as well as among the all
accessions. These results suggested that higher genetic diversity could
be detected by ISSR markers than that of RAPD markers among the 46
barley accessions from west China.

The relationships within and between groups were estimated by a UPGMA
cluster analysis of GS matrices (Fig 1.). It indicated that all 46
barley accessions could be distinguished by both RAPD and ISSR markers.
Fig. 1a was the dendrogram based on RAPD data. One HV accession (B18)
with six rowed and hulled, which collected from Hongyuan, Sichuan, was
less related with other genotypes, and divergent from the others. Two
subgroups were evident for the remaining 45 genotypes. The first
subgroup contained 17 accessions, including 2 HS accessions (ZYM0059 and
ZYM0083) and 15 HV accessions. The second subgroup consisted of 28
accessions, including 4 HS accession, 13 HA accessions and 11 HV
accessions. The genotypes were more closely related with each other
within the second subgroup, whereas more diverse within the first
subgroup.

Fig. 1b was the dendrogram generated from ISSR data. One HA accession
(ZYM0834) and One HV accession ( B118), which were collected from
Xizang, were less related with other genotypes, and divergent from the
others. Two subgroups were evident for the remaining 44 accessions, with
the first subgroup only including one HS accession (ZYM0059) and two HA
accessions (ZYM0830 and ZYM0469). The second subgroup including 41
genotypes consisted of 5 HS accessions, 10 HA accessions and 26 HV
accessions. The genotypes were more closely related with each other
within the second subgroup, while more diverse within the first
subgroup.

The correlation coefficient for the elements of the RAPD-GS and ISSR-GS
matrices was calculated using the Mantel test (Mantel 1967). There was
no significant correlation (r = 0.113) between the RAPD-GS and ISSR-GS
matrices, indicating that both sets of markers revealed the unrelated
estimates of genetic relationships.

# 

# Discussion

In this study, genomic DNA was extracted from a bulk sampling for each
accession. The advantages and inconvenient of the bulk analysis have
been discussed by Michelmore et al. (1991) and Loarce et al. (1996).
Bulk analyses are economic and rapid, and it is possible to estimate the
genetic variability between accessions, whereas it is not possible to
obtain information about the genetic variability within the accessions
(Fernández et al. 2002). The number of individual plants bulked for the
accessions is an important experimental factor whether the bulked
analysis revealed the genetic relationship between the accessions. Yang
and Quiros (1993) found that the bulked samples with 10, 20, 30, 40 and
50 individuals had the same banding pattern. Bustos et al. (1998) also
found that bulks of 10 to 20 individuals resulted in the same RAPD
profiles. In this study, we used a minimum of ten individuals for
representing the each barley accession. The results indicated that the
percentage of ISSR polymorphic bands (98.13%) was higher than that of
RAPD (77.06%). The mean number of amplification RAPD bands (4.19) was
more than that of ISSR (5.94). Moreover, the total number of polymorphic
bands (105) detected by 18 ISSR primers was much higher than that of the
26 RAPD primers (84). And the mean polymorphism information content
(PIC) (0.631) in ISSR analysis also was higher than that in RAPD
analysis (0.574). The bulk analysis of RAPD and ISSR markers could
successfully be used to investigate the genetic diversity of the barley
landraces and the wild forms from west China. However, the results in
this study suggested that the ISSR markers were superior to RAPD markers
in the capacity of revealing more informative bands in a single
amplification. The similar results were observed by Fernández et al.
(2002) and Tanyolac (2003).

Due to its worldwide distribution, the valuation of the genetic
diversity among barley germplasm from different countries has been
performed (Tanyolac 2003; Liu et al. 2002; Fernández et al. 2002; Matus
et al. 2002; Dávila et al. 1999a, 1999b; Dávila et al. 1998; Konishi
2001; Bustos et al. 1998; Bjornstad et al. 1997; Chen et al. 2000b; Feng
et al. 2003). Bernard et al. (1997) analyzed the genetic diversity in 88
genotypes from 20 populations of wild barley from Israel, Turkey and
Iran by RAPD markers. When the total genetic diversity was estimated,
75% of the variation detected was partitioned within the 88 genotypes
and 25% among the populations. When variation between countries was
assessed, no substantial differences were found, because most of the
variation detected (97%) was partitioned within the 20 populations and
the remainder among the countries. Russell et al. (1997) found that the
average genetic diversity based on RAPD analyses of eighteen accessions
from Netherlands, France, Great Britain, Germany and Italy was 0.521.
Bahattin (2003) assayed 15 wild barley populations from west Turkey by
using RAPD and ISSR markers. The results revealed that the average
genetic similarity was 0.27 and the genetic variation was higher than
that found by Nevo et al. (1979) and Nevo et al. (1986) both using
isozyme markers. In the present study, the average genetic similarities
of barley accessions from western China based on RAPD and ISSR markers
were 0.864 and 0.674, respectively. The similarities detected with RAPDs
are greater than the similarities observed with ISSRs. Fernández et al.
(2002) also found similar results. The genetic variation found in this
study was equivalent with that found by Chen et al. (2000a) (0.81), Shi
et al. (2004) (0.631) and Chen et al. (2000b) (0.746) both using RAPD
markers to analyze the genetic variation of different barley populations
from China. But the variation was relatively lower than that from other
country (Russell et al. 1997; Bahattin 2003).

In this study, it was obvious that the dendrogram based on RAPD markers
was not in accord with the dendrogram based on ISSR markers. The
dendrogram generated by the RAPD matrix agrees better with the groups of
the genotypes than the dendrogram generated by the ISSR results (Fig.
1a). Most of the genotypes within the wild barley and the landrace were
closely related. However, the dendrogram generated by the ISSR matrix
agrees better with the geographic origins of the genotypes than the
dendrogram generated by the RAPD results (Fig. 1b). For example, three
landraces (i.e. B65, B118 and B120) originating from Xizang, were
closely clustered with the wild forms originating from Xizang. Fernández
et al. (2002) found the dendrogram generated by the ISSR matrix agrees
better with the genealogy and the known pedigree of the barley cultivars
than the dendrogram generated by the RAPD results. Wu et al. (2004)
found that the data based on RAPD-GS were more correlated with the
geographic distribution of the genus *Houttuynia Thunb*, while the data
based on ISSRs were closely related with their number of chromosomes. It
could be partially explained by the different number of informative PCR
products (84 for RAPDs and 105 for ISSRs). They reinforced again the
importance of the number of loci and their coverage of the overall
genome and obtained reliable estimates of genetic relationship among the
studied materials (Fernández et al. 2002). Similar results had been
observed by Loarce et al. (1996). On the other hand, the relationship
observed using molecular markers may provide information on the history
and biology of cultivars, but it does not necessarily reflect what may
be observed with respect to agronomic traits (Métais et al. 2000). The
selection process leads to an accumulation of best alleles for the
traits under selection. RAPDs and ISSRs are dispersed throughout the
genome and their association with agronomic traits is influenced by the
breeder only in the region under selection pressure. The other loci are
subjected to random genetic drift (Fernández et al. 2002). Another
explanation could be the putatively similar bands originating for RAPDs
in different bulked samples were not necessarily homologous although
they shared the same size in base pairs (Karp et al. 1997). This
situation might lead to wrong results when calculating genetic
relationships (Fernández et al. 2002).

# 

# Acknowledgments

The wild barley accessions were kindly supplied by Prof. D. Q. Ma
(Institute of Crop Germplasm Resources, CAAS). This work was supported
by the Hi-Tech Research and Development (863) Program of China
(2003AA207100) and the Foundation for the Author of National Excellent
Doctoral Dissertation of PR China (200357). Y.-L. Zheng was supported by
Program for Changjiang Scholars and Innovative Research Team in
University (PCSIRT), China.

# 

# References

Asins MJ, EA Carbonell. 1989. Distribution of genetic variability in a
durum wheat world collection. Theor Appl Genet., 77: 287-294

Akkaya MS, AA Bhagwast and PB Cregan. 1992. Length polymorphisms of
simple sequence repeat DNA in soybean. Genetics., 132: 1131-1139

Bahattin T. 2003. Inter-simple sequence repeat (ISSR) and RAPD variation
among wild barley (*Hordeum vulgare* subsp. *spontaneum*) populations
from west Turkey. Genetic Resources and Crop Evolution., 50: 611-614

Barua UM, KJ Chalmers, CA Hackett, WTB Thomas, W Powell, R Waugh. 1993.
identification of RAPD markers linked to a phynchosporium secalis
resistance locus in barley using nearisogenic lines and bulked segregant
analysis. Heredity., 71: 177-184

Bernard RB, E Nevo, AJ Douglas and A Beiles. 1997. Genetic diversity in
wild barley (*Hordeum spontaneum* C. Koch) in the near east: a molecular
analysis using random amplified polymorphic DNA (RAPD) markers. Genetic
Resources and Crop Evolution., 44: 147-157

Bustos AD, C Casanova, C Soler, N Jouve. 1998. RAPD variation in wild
populations of four species of the genus *Hordeum* (*Poaceae*). Theor
Appl Genet., 96: 101-111

Brown ADH, JJ Burdon, JP Grace. 1990. Genetic structure of *Glycine
canescens*. A perennial relative of soybean. Theor Appl Genet., 79:
729-736

Bjornstad A, A Demisse, A Killian, A Kleinhofs. 1997. The distinctness
and diversity of Ethiopian barley. Theor Appl Genet., 94: 514-521

Botstein D, RL White, M Skolnick, RW Davis. 1980. Construction of a
genetic linkage map in man using restriction fragment length
polymorphisms. Am. J. Hum. Genet.,32: 314-331

Chen XP, L Yan, Y Ding, WX Gao. 2000a. Analysis of germplasms of Hubei
barley by randomly amplified polymorphic DNA (RAPD). J. Wuhan Univ.
(Natural Science Edition)., 46(2): 249-252

Chen XP, L Yan, Y Ding. 2000b. RAPD analysis and the probable
evolutionary route of wild relatives of barley from China. Acta Botanica
Sinica., 42(2): 179-183

Chalmers KJ, UM Barua, CA Hackett, WTB Thomas, R Waugh, W Powell. 1993.
Identification of RAPD markers linked to genetic factors controlling the
milling energy requirement of barley. Theor Appl Genet., 87: 314-320

Dávila JA, Y Loarce, L Ramsay, R Waugh, E Ferrer. 1999a. Comparison of
RAMP and SSR markers for the study of wild barley genetic diversity.
Hereditas., 131: 5-13

Dávila JA, Y Loarce, E Ferrer. 1999b. Molecular characterization and
genetic mapping of random amplified microsatellite polymorphism in
barley. Theor Appl Genet., 98: 265-273

Dávila JA, MP Sánchez de la Hoz, Y Loarce, E Ferrer. 1998. The use of
random amplified microsatellite polymorphic DNA and coefficients of
parentage to determine genetic relationship in barley. Genome., 41:
477-486

Devos KM, MD Gale. 1992. The use of random amplified polymorphic DNA
markers in wheat. Theor Appl Genet., 84: 567-572

Fang DQ, ML Roose, RR Krueger, CT Federicé. 1997. Fingerprinting
trifoliate orange germplasm accessions with isozymes, RFLP and
inter-simple sequence repeat markers. Theor Appl Genet., 95: 211-219

Feng ZY, YZ Zhang, LL Zhang, HQ Ling. 2003. Genetic diversity and
geographical differentiation of *Hordeum vulgare* ssp. *spontaneum* in
Tibet using microsatellite markers. High Technology Letters., 10: 46-53

Fernández ME, AM Figueiras, C Benito. 2002. The use of ISSR and RAPD
markers for detecting DNA polymorphism, genotype identification and
genetic diversity among barley cultivars with known origin. Theor Appl
Genet., 104: 845-851

Hong QB, L Hou, XY Luo, DM Li, YH Xiao, Y Fei, KJ Yang, C Jia. 2001.
Using RAPD for evaluating genetic background among naked barley
varieties in Sichuan northwestern region. Sci Agric Sin., 34(2): 133-138

Joshi Cp, HT Nguyen. 1993. RAPD (Random amplified polymorphic DNA)
analysis based on intervarietal genetic relationships among hexaploid
wheats. Plant Sci., 93: 95-103

Karp A, K Edwards, M Bruford. 1997. Newer molecular technologies for
biodiversity evaluation: opportunities and challenges. Nature
Biotechnol., 15: 625-628

Kantety RV, XP Zeng, JL Bennetzen, BE Zehr. 1995. Assessment of genetic
diversity in Dent and Popcom (Zea mays L.) inbred lines using
inter-simple sequences repeat (ISSR) amplification. Mol Breed., 1:
365-373

Konishi T. 2001. Genetic diversity in *Hordeum agriocrithon* E. Åberg,
six-rowed barley with brittle rachis, from Tibet. Genetic Resource and
Crop Evolution., 48: 27-34

Loarce Y, R Gallego, E Ferrer. 1996. A comparative analysis of genetic
relationship between rye cultivars using RFLP and RAPD markers.
Euphytica., 88: 107-115

Liu F, GL Sun, B Salomon, R Von Bothmer. 2002. Characterization of
genetic diversity in core collection accessions of wild barley, *Hordeum
vulgare* ssp. *spontaneum*. Hereditas., 136: 67-73

Martín JP, MD Sánchez-Yélamo. 2000. Genetic relationships among species
of the genus *Diplotaxis* (*Brassicaceae*) using inter-simple sequence
repeat markers. Theor Appl Genet., 101: 1234-1241

Matus IA and PM Hayes. 2002. Genetic diversity in three groups of barley
germplasm assessed by simple sequence repeats. Genome., 45: 1095-1106

Mantel M. 1967. The detection of disease clustering and a generalized
regression approach. Cancer. Res., 27: 209-220

Métais I, C Aubry, B Hamon, R Jalouzot. 2000. Description and analysis
of genetic diversity between commercial bean lines (*Phaseolus vulgaris*
L.). Theor Appl Genet., 101: 1207-1214

Michelmore RW, I Paran, RV Kesseli. 1991. Identification of markers
linked to disease resistance genes by bulk segregant analysis: a rapid
method to detect markers in specific genomic regions using segregating
populations. Proc Natl Acad Sci., USA. 88: 9828-9832

Nevo E, D Zohary, AHD Brown and M Haber. 1979. Genetic diversity and
environmental associations of wild barley, *Hordeum spontaneum*, in
Israel. Evolution., 33: 815-833

Nevo E, D Zohary, A Beiles, D Kaplan and N Storch. 1986. Genetic
diversity and environmental associations of wild barley, *Hordeum
spontaneum*, in Turkey. Genetica., 68: 203-213

Nevo E. 1992. Origin, evolution, population genetic and resources for
breeding of wild barley. *Hordeum spontaneum*. In the fertile Crescent.
In: Barley: Genetics. Biochemistry. Molecular Biology and Biotechnology
(ed PR Shewry) CAB international. Wallingford. 19-143

Nagaoka T and Y Ogihara. 1997. Applicability of inter-simple sequence
repeat polymorphism in wheat for use as DNA markers in comparison to
RFLP and RAPD markers. Theor Appl Genet., 94: 597-602

Nei M, W Li. 1979. Mathematical model for studying genetic variation in
terms of restriction endonucleases. Proc. Natl. Acad. USA., 76:
5269-5273

Plucknett DL, NJH Smith, JT Williams, AN Murthi. 1987. Genebanks and the
world's food. Princeton University Press, Princeton New Jersey, USA.

Pejic I, P Ajmone-Marsan, M Morgante, V Kozumplick, P Castiglioni, G
Taramino, M Motto. 1998. Comparative analysis of genetic similarity
among maize inbred lines detected by RFLPs, RAPDs, SSRs and AFLPs. Theor
Appl Genet., 97: 1248-1255

Prevost A, MJ Wikinson. 1999. A new system of comparing PCR primers
applied to ISSR fingerprinting of potato cultivars. Theor Appl Genet.,
98: 107-112

Powell W, M Morgante, C Andre. 1996. The comparision of RFLP, RAPD, AFLP
and SSR(microsatellite) marker for germplasm analysis. Mol Breeding., 2:
225-238

Qian W, S Ge and DY Houng. 2001. Genetic variation within and among
populations of a wild rice Oryza granulata from China detected by RAPD
and ISSR markers. Thore Appl Genet., 102: 440-449

Rohlf FJ. 1993. NTSYS-pc version 1.80. Distribution by Exeter Software,
Setauket, New York.

Russell JR, JD Fuller, M Macaulay, BG Hatz, A Jahoor, W Powell, R Waugh.
1997. Direct comparison of levels of genetic variation among barley
accessions detected by RFLPs, AFLPs, SSRs and RAPDs. Theor Appl Genet.,
95: 714-722

Sokal RR and FJ Rohlf. 1995. Biometry: The principles and practice of
statistics in biological research. (3d edition). WH Freeman and Company.
New York. USA.

Saiki RK, DH Gelfond, S Stoffel, SJ Scharf, R Higuchi, BT Horn, KB
Mullis and HA Erlich. 1988. Primer-directed anzymatic amplification of
DNA with a thermostable DNA polymerase. Science., 239: 487-491

Sánchez de la Hoz MP, JA Dávila, Y Loarce and E Ferrer. 1996. Simple
sequence repeat primers used in polymerase chain reaction amplifications
to study genetic diversity in barley. Genome., 39: 112-117

Struss D and J Plieske. 1998. The use of microsatellite markers for
detection of genetic diversity in barley populations. Theor Appl Genet.,
97: 308-315

Shi YT, HW Bian, N Han, JW Pan, WX Tong, MY Zhu. 2004. Genetic variation
analysis by RAPD of some barley cultivars in China. Acta Agronomica
Sinica., 30(3): 258-265

Sharp PJ, M Kresis, PR Shewry and MD Gale. 1988. Location of β-amylase
sequences in wheat and its relatives. Theor Appl Genet., 75: 286-290

Sneath PHA and RR Sokal. 1973. Numerical Taxonomy. Freeman, San
Francisco.

Tragoonrung S, V Kanazin, PM Hayes and TK Blake. 1992.
Sequence-tagged-site facilitated PCR for barley genome mapping. Theor
Appl Genet., 84: 1002-1008

Tanyolac B. 2003. Inter-simple sequence repeat (ISSR) and RAPD variation
among wild barley (Hordeum vulgare subsp. spontaneum) populations from
west Turkey. Genetic Resources and Crop Evolution., 50: 611-614

Tinker NA, MG Fortin, DE Mather. 1993. Random amplified polymorphic DNA
and pedigree relationship in spring barley. Theor Appl Genet., 85:
976-984

von Bothmer R, N Jacobsen, C Baden, RB Jørgensen and I Linde-Laursen.
1995. An ecogeographical study of the genus *Hordeum*. International
Plant Genetic Resources, Rome. P.29.

Welsh J and J McClelland. 1990. Fingerprinting genomes using PCR with
arbitrary primers. Nucleic Acids Res., 18: 7213-7218

Williams JGK, AR Kubelik, KJ Livak, JA Rafalski and SV Tingey. 1990. DNA
polymorphisms amplified by arbitrary primers are useful as genetic
markers. Nucleic Acids Res., 18: 6531-6535

Wu KS, J R ones, L Danneberger. 1994. Detection of microsatellite
polymorphisms without cloning. Nucleic Acids Res., 22: 3257-3258

Wu W，YL Zheng，L Chen， YM Wei，RW Yang and ZH Yan. 2005. Evaluation of
genetic relationships in the genus *Houttuynia Thunb*. in China based on
RAPD and ISSR markers. Biochemical Systematics and Ecology., (Accepted)

Yang JL, C Yen, BR Lu, R von Bothmer. 1987. The genus *Hordeum*
(Gramineae) in China. Variation and distribution. Willdenowia, 16:
479-490

Yang X, C Quiros. 1993. Identification and classification of celery
cultivars with RAPD markers. Theor Appl Genet., 86: 205-212

Zietkiewicz E, A Rafalski, D Labuda. 1994. Genome fingerprinting by
simple sequence reprat (SSR)-anchored polymerase chain reaction
amplification. Genomics.,20: 176-783

Table 1. Origin, group and rowed type of 46 landraces and wild barley
accessions form west China

<table>
<colgroup>
<col style="width: 8%" />
<col style="width: 5%" />
<col style="width: 4%" />
<col style="width: 7%" />
<col style="width: 15%" />
<col style="width: 1%" />
<col style="width: 8%" />
<col style="width: 7%" />
<col style="width: 7%" />
<col style="width: 10%" />
<col style="width: 21%" />
</colgroup>
<tbody>
<tr class="odd">
<td>Acc.no.</td>
<td>Group</td>
<td><p>Row</p>
<p>type</p></td>
<td><p>Hulled or</p>
<p>Naked</p></td>
<td>Origin</td>
<td></td>
<td><p>Acc.</p>
<p>no.</p></td>
<td>Group</td>
<td>Row type</td>
<td><p>Hulled or</p>
<p>Naked</p></td>
<td>Origin</td>
</tr>
<tr class="even">
<td>ZYM0022</td>
<td>HS</td>
<td>Two</td>
<td>Hulled</td>
<td>Gongbujiangda, Xizang</td>
<td></td>
<td>B20</td>
<td>HV</td>
<td>Six</td>
<td>Naked</td>
<td>Mianning, Sichuan</td>
</tr>
<tr class="odd">
<td>ZYM0059</td>
<td>HS</td>
<td>Two</td>
<td>Hulled</td>
<td>Longzi, Xizang</td>
<td></td>
<td>B21</td>
<td>HV</td>
<td>Six</td>
<td>Naked</td>
<td>Ma’erkang, Sichuan</td>
</tr>
<tr class="even">
<td>ZYM0083</td>
<td>HS</td>
<td>Two</td>
<td>Hulled</td>
<td>Cuola, Xizang</td>
<td></td>
<td>B22</td>
<td>HV</td>
<td>Six</td>
<td>Naked</td>
<td>Dangxiong, Xizang</td>
</tr>
<tr class="odd">
<td>ZYM0182</td>
<td>HS</td>
<td>Two</td>
<td>Hulled</td>
<td>Qusong, Xizang</td>
<td></td>
<td>B23</td>
<td>HV</td>
<td>Six</td>
<td>Naked</td>
<td>Minshan, Gansu</td>
</tr>
<tr class="even">
<td>ZYM0195</td>
<td>HS</td>
<td>Two</td>
<td>Hulled</td>
<td>Qiongjie, Xizang</td>
<td></td>
<td>B33</td>
<td>HV</td>
<td>Six</td>
<td>Naked</td>
<td>Xiahe, Gansu</td>
</tr>
<tr class="odd">
<td>ZYM0212</td>
<td>HS</td>
<td>Two</td>
<td>Hulled</td>
<td>Dingri, Xizang</td>
<td></td>
<td>B40</td>
<td>HV</td>
<td>Six</td>
<td>Naked</td>
<td>Li, Sichuan</td>
</tr>
<tr class="even">
<td>ZYM0281</td>
<td>HA</td>
<td>Six</td>
<td>Hulled</td>
<td>Luolong, Xizang</td>
<td></td>
<td>B47</td>
<td>HV</td>
<td>Six</td>
<td>Naked</td>
<td>Ganzi, Sichuan</td>
</tr>
<tr class="odd">
<td>ZYM0310</td>
<td>HA</td>
<td>Six</td>
<td>Hulled</td>
<td>Nimu, Xizang</td>
<td></td>
<td>B53</td>
<td>HV</td>
<td>Six</td>
<td>Naked</td>
<td>Jinchuan, Sichuan</td>
</tr>
<tr class="even">
<td>ZYM0315</td>
<td>HA</td>
<td>Six</td>
<td>Hulled</td>
<td>Mozhugongka, Xizang</td>
<td></td>
<td>B65</td>
<td>HV</td>
<td>Six</td>
<td>Naked</td>
<td>Lasa, Xizang</td>
</tr>
<tr class="odd">
<td>ZYM0407</td>
<td>HA</td>
<td>Six</td>
<td>Hulled</td>
<td>Luozha, Xizang</td>
<td></td>
<td>B76</td>
<td>HV</td>
<td>Six</td>
<td>Hulled</td>
<td>Lu, Sichuan</td>
</tr>
<tr class="even">
<td>ZYM0444</td>
<td>HA</td>
<td>Six</td>
<td>Hulled</td>
<td>Naidong, Xizang</td>
<td></td>
<td>B77</td>
<td>HV</td>
<td>Six</td>
<td>Hulled</td>
<td>Peng’an, Sichuan</td>
</tr>
<tr class="odd">
<td>ZYM0469</td>
<td>HA</td>
<td>Six</td>
<td>Hulled</td>
<td>Rikaze, Xizang</td>
<td></td>
<td>B83</td>
<td>HV</td>
<td>Four</td>
<td>Naked</td>
<td>Wenchuan, Sichuan</td>
</tr>
<tr class="even">
<td>ZYM0494</td>
<td>HA</td>
<td>Six</td>
<td>Hulled</td>
<td>Zhada, Xizang</td>
<td></td>
<td>B87</td>
<td>HV</td>
<td>Six</td>
<td>Naked</td>
<td>Xiaojin, Sichuan</td>
</tr>
<tr class="odd">
<td>ZYM0531</td>
<td>HA</td>
<td>Six</td>
<td>Hulled</td>
<td>Luolong, Xizang</td>
<td></td>
<td>B95</td>
<td>HV</td>
<td>Six</td>
<td>Hulled</td>
<td>Miyi, Sichuan</td>
</tr>
<tr class="even">
<td>ZYM0545</td>
<td>HA</td>
<td>Six</td>
<td>Hulled</td>
<td>Chaya, Xizang</td>
<td></td>
<td>B96</td>
<td>HV</td>
<td>Four</td>
<td>Hulled</td>
<td>Santai, Sichuan</td>
</tr>
<tr class="odd">
<td>ZYM0606</td>
<td>HA</td>
<td>Six</td>
<td>Hulled</td>
<td>Milin, Xizang</td>
<td></td>
<td>B97</td>
<td>HV</td>
<td>Six</td>
<td>Naked</td>
<td>Ruo’ergai, Sichuan</td>
</tr>
<tr class="even">
<td>ZYM0642</td>
<td>HA</td>
<td>Six</td>
<td>Hulled</td>
<td>Linzhou, Xizang</td>
<td></td>
<td>B98</td>
<td>HV</td>
<td>Four</td>
<td>Hulled</td>
<td>Nanchong, Sichuan</td>
</tr>
<tr class="odd">
<td>ZYM0830</td>
<td>HA</td>
<td>Six</td>
<td>Hulled</td>
<td>Jiangzi, Xizang</td>
<td></td>
<td>B108</td>
<td>HV</td>
<td>Six</td>
<td>Naked</td>
<td>Heishui, Sichuan</td>
</tr>
<tr class="even">
<td>ZYM0834</td>
<td>HA</td>
<td>Six</td>
<td>Hulled</td>
<td>Renbu, Xizang</td>
<td></td>
<td>B115</td>
<td>HV</td>
<td>Six</td>
<td>Naked</td>
<td>Nanping, Sichuan</td>
</tr>
<tr class="odd">
<td>B4</td>
<td>HV</td>
<td>Six</td>
<td>Naked</td>
<td>Ruo’ergai, Sichuan</td>
<td></td>
<td>B118</td>
<td>HV</td>
<td>Six</td>
<td>Naked</td>
<td>Gongka, Xizang</td>
</tr>
<tr class="even">
<td>B10</td>
<td>HV</td>
<td>Six</td>
<td>Naked</td>
<td>Rangtang, Sichuan</td>
<td></td>
<td>B120</td>
<td>HV</td>
<td>Six</td>
<td>Naked</td>
<td>Mozhugongka, Xizang</td>
</tr>
<tr class="odd">
<td>B12</td>
<td>HV</td>
<td>Six</td>
<td>Naked</td>
<td>Songpan, Sichuan</td>
<td></td>
<td>B124</td>
<td>HV</td>
<td>Six</td>
<td>Naked</td>
<td>Qingdao</td>
</tr>
<tr class="even">
<td>B18</td>
<td>HV</td>
<td>Six</td>
<td>Naked</td>
<td>Hongyuan, Sichuan</td>
<td></td>
<td>B125</td>
<td>HV</td>
<td>Six</td>
<td>Hulled</td>
<td>Xichang, Sichuan</td>
</tr>
</tbody>
</table>

Table 2. Primer sequences, amplified bands, polymorphic bands and PIC
values in RAPD analysis

  --------- ----------------- ----------------- ------------------- -------
  Primer    Sequence (5'-3')  Amplified bands   Polymorphic bands   PIC

  OPA-1     CAGGCCCTTC        4                 3                   0.677

  OPA-4     AATCGGGCTG        2                 2                   0.639

  OPA-11    CAATCGCCGT        5                 4                   0.647

  OPA-13    CAGCACCCAC        4                 2                   0.442

  OPA-18    AGGTGACCGT        6                 6                   0.316

  OPB-1     GTTTCGCTCC        3                 2                   0.453

  OPB-2     TGATCCCTGG        5                 4                   0.412

  OPB-3     CATCCCCCTG        7                 6                   0.603

  OPB-10    CTGCTGGGAC        7                 3                   0.762

  OPB-12    CCTTGACGCA        7                 6                   0.293

  OPB-18    CCACAGCAGT        4                 1                   0.360

  OPB-20    GGACCCTTAC        8                 8                   0.550

  OPH-7     CTGCATCGTG        4                 4                   0.619

  OPH-12    ACGCGCATGT        5                 5                   0.894

  OPH-13    GACGCCACAC        4                 4                   0.156

  OPH-18    GAATCGGCCA        5                 3                   0.744

  OPH-19    CTGACCAGCC        3                 2                   0.407

  OPP-3     CTGATACGCC        4                 2                   0.486

  OPP-5     CCCCGGTAAC        2                 1                   0.741

  OPP-8     ACATCGCCCA        3                 2                   0.816

  OPP-9     GTGGTCCGCA        2                 1                   0.753

  OPP-11    AACGCGTCGG        2                 2                   0.724

  OPP-12    AAGGGCGAGT        2                 2                   0.708

  OPP-14    CCAGCCGAAC        3                 3                   0.659

  OPP-16    CCAAGCTGCC        4                 3                   0.439

  OPP-17    TGACCCGCCT        4                 3                   0.623

  Total                       109               84                  

  Mean                        4.19              3.23                0.574
  --------- ----------------- ----------------- ------------------- -------

Table 3. Primer sequences, amplified bands, polymorphic bands and PIC
values in ISSR analysis

  --------- ------------------ ---------------- ------------------- -------
  Primer    Sequence (5'-3')\* Amplified bands  Polymorphic bands   PIC

  ISSR-1    HVH(CA)~7~T        6                6                   0.516

  ISSR-2    (AG)~8~C           4                4                   0.618

  ISSR-3    (GA)~8~T           5                5                   0.763

  ISSR-4    (CT)~8~G           7                7                   0.693

  ISSR-5    (CA)~6~ACAG        8                8                   0.645

  ISSR-6    BDB(TCC)~5~        2                2                   0.816

  ISSR-7    HVH(TCC)~5~        2                2                   0.835

  ISSR-8    DBDA(CA)~7~        6                6                   0.808

  ISSR-9    (CA)~8~RG          5                5                   0.533

  ISSR-10   (GA)~8~YG          6                6                   0.396

  ISSR-11   (AC)~8~YG          10               10                  0.509

  ISSR-12   (AG)~8~YT          3                3                   0.723

  ISSR-13   (AC)~8~C           7                7                   0.590

  ISSR-14   (GA)~8~YC          3                3                   0.760

  ISSR-15   (GA)~8~C           8                7                   0.455

  ISSR-16   (AC)~8~YA          7                7                   0.351

  ISSR-17   (TC)~8~C           8                8                   0.478

  ISSR-18   (TG)~8~G           10               9                   0.874

  Total                        107              105                 

  Mean                         5.94             5.83                0.636
  --------- ------------------ ---------------- ------------------- -------

\* R=A/T, Y=G/C, B=T/G/C; D=A/T/G, H=A/T/C, V=A/G/C

Table 4. RAPD-and ISSR-based genetic similarity within groups.

  -------- ----------- ------------- ------------- ------------- -------------
  Group                HV            HS            HA            All

  RAPD     Mean        0.867         0.895         0.877         0.864

           Variation   0.763-0.980   0.849-0.954   0.800-0.954   0.735-0.980

  ISSR     Mean        0.716         0.645         0.623         0.674

           Variation   0.350-0.931   0.342-0.832   0.250-0.911   0.212-0.931
  -------- ----------- ------------- ------------- ------------- -------------

![](media/image2.jpeg){width="6.2652777777777775in"
height="4.507638888888889in"}

![](media/image3.jpeg){width="6.263888888888889in" height="4.425in"}

Fig. 1 Dendrograms of 46 barley accessions constructed from RAPD-GS (a)
and ISSR- GS (b) matrices