Theor Appl Genet (1998) 97 : 515—519 ( Springer-Verlag 1998

M. B. Ratnaparkhe · M. Tekeoglu · F. J. Muehlbauer

Inter-simple-sequence-repeat (ISSR) polymorphisms are useful for finding markers

associated with disease resistance gene clusters

Received: 20 January 1998 / Accepted: 19 March 1998

Abstract We describe a simple and new approach, Key words Microsatellite · Disease resistance ·
based on inter-simple sequence repeats (ISSRs), for Mapping · Sequence-directed approach
finding markers linked to clusters of disease resistance
genes. In this approach, simple sequence repeats (SSR)
are used directly in PCR reactions, and markers found
to be linked to disease resistance genes provide impor- Introduction
tant information for the selection of other sequences
which can be used with PCR to find other linked Inter-simple sequence repeats (ISSRs) are a new type of
markers. Based on an ISSR marker linked to a gene of DNA marker which involves the use of microsatellite
interest, many new markers can be identified in the sequences directly in the polymerase chain reaction
same region. We previously demonstrated that ISSR (PCR) for DNA amplifications (Gupta et al. 1994; Ziet-
markers are useful in gene tagging and identified kiewicz et al. 1994; Sanchez et al. 1996). This technique
a marker, UBC-855 , linked to the gene for resistance enables amplification of genomic DNA and provides
500
to fusarium wilt race 4 in chickpea. This ISSR marker information about many loci simultaneously. Recent
provided the information used in the present study for reports have shown Mendelian inheritance of simple-
selecting other primers which amplified a region linked sequence-repeat (SSR) fragments scored as dominant
to the gene for resistance to fusarium wilt race 4. The markers (Tsumura et al. 1996) or as codominant
primers were based on homology with the (AC) se- markers (Wu et al. 1994). Simple sequence repeats
/
quence and were used for PCR amplifications. Changes (SSRs) are short tandem repetitive DNA sequences
in the sequence were at the anchor region of the with a repeat length of few base pairs (1-5) (Litt and
primers. The repeat (AC) T amplified a marker, UBC- Luty 1989). The sequences are abundant, dispersed
8
825 , which was located 5.0 cM from the gene for throughout the genome and highly polymorphic in
1200
resistance to fusarium wilt race 4 and was closer than comparison with other molecular markers (Akkaya et
other markers. These results indicated that ISSR al. 1992; Morgante and Olivieri 1993; Wang et al. 1994).
markers can provide important information for the SSRs have been used successfully in the genome map-
design of other primers and that by making changes at ping of a variety of crop species including maize, rice,
the 3@ and 5@ anchors close linkage to the desired gene barley and wheat (Senior and Heun 1993; Wu and
can be found. The approach allows rapid scanning of Tanksley 1993; Saghai-Maroof et al. 1994; Roder et al.
the targeted region and may provide important in- 1995). However, due to the technical difficulties in de-
formation for genome analysis of plant species. veloping SSRs, these markers have not been commonly
used for gene tagging in plants. ISSR has been pro-
posed as a new source of genetic markers which over-
comes the technical limitations of restriction fragment
length polymorphism (RFLP) and random amplified
Communicated by P. M. A. Tigerstedt
polymorphic DNA (RAPD).
Ratnaparkhe et al. (1998) were the first to demon-
M. B. Ratnaparkhe · M. Tekeoglu · F. J. Muehlbauer ( ) strate that ISSR markers are useful in gene tagging and
USDA-ARS, Grain Legume Genetics and Physiology Unit,
and Department of Crop and Soil Sciences, can be used for finding markers linked to the gene of
Washington State University, Pullman WA 99164, USA interest. Previous reports indicated that SSRs are
Fax: #1 509-335-7692 not randomly distributed in the genome but are often
516

clustered (Arens et al. 1995; Broun and Tanksley 1996). described by Tullu (1996). When the filter paper was completely
The clustering of SSRs has also been observed in hu- colonized by fungus, only those colonies representative of the wild
mans (Erickson et al. 1988) and on sex chromosomes of type were aseptically removed and placed in a fresh petri dish to dry
for 5 days in a laminar flow hood. The dried filter paper was
different organisms (Epplan 1988; Nanda et al. 1990). aseptically cut into pieces using a pair of sterile scissors, and these
Similarly, disease resistance genes have also been found pieces were then used to prepare the primary inoculum. The conidia
to form clusters (Sheperd and Mayo 1972; Islam et al. concentration was adjusted to 1 ] 106 spores per milliliter with
1993; Kesseli et al. 1993). Studies on disease resistance a hemacytometer. Twelve to twenty seeds of each RIL were grown in
the greenhouse (21°—26°C) in single rows in plastic trays filled with
genes have indicated a high level of polymorphism and sterile coarse perlite. When the seedlings reached the three- to
the presence of SSRs at certain loci (Yu et al. 1996). In four-nodal stage, they were carefully removed from the perlite,
the study presented here we exploited the combination pruned while submerged in the spore suspension and, after about
of ISSR markers to find markers at the fusarium wilt 5 min in the spore suspension, replanted into the perlite. Plants were
disease resistance gene cluster. The approach is based then scored as susceptible or resistant over the next 2 months.
on the association of a cluster of SSRs with the disease
resistance gene cluster. We also investigated the poten-
tial of an ISSR-directed approach for selecting other Oligonucleotide primers
sequences which can be used to find linked markers
One hundred primers of 15—23 nucleotides in length (UBC set K9)
and for marker enrichment in the desired region. The were obtained from the Biotechnology Laboratory, University of
technique is based on the use of SSR primers with British Columbia, Vancouver, British Columbia, Canada and used
variations at 5@ and 3@ anchors, which provides markers for polymerase chain reaction (PCR) amplifications.
linked to the desired gene. We studied the inheritance
of ISSR polymorphisms using 96 recombinant inbred
lines (RILs) derived from the cross of C. arietinum PCR amplification and electrophoresis
(ICC-4958), a cultivated chickpea germplasm line
with resistance to fusarium wilt, and C. reticulatum PCR amplification was performed in 10 mM TRIS-HCl pH 8.3,
(PI 489777), the closest wild relative of the cultivated 50 mM KCl, 0.1% Triton ]100, 2.5 mM MgCl , 0.2 lM dNTP,
2
0.24 l, of primer, 30 ng of genomic DNA per 25 ll of reaction
species, and identified markers linked to the gene for volume and 1 unit of ¹aq polymerase. The amplifications were
resistance to fusarium wilt races 4 and 5. carried out on a Perkin Elmer Cetus 9600 programmed for 35 cycles
of de-naturation at 94°C for 30 s, annealing at 50°C for 30 s and
extension at 72°C for 2 min, with a 10-min final extension at 72°C.
Materials and methods PCR products were separated on 2% agarose gels, then stained with
ethidium bromide and scored for presence or absence of bands.
Since ISSR markers are dominant, a locus was considered to be
Plant material
polymorphic if the band was present in one parent and not in the
other. Linkage analysis was performed using the MAPMAKER
A set of 96 F -derived F recombinant inbred lines (RILs) obtained
6 7 program (Lander et al. 1987)
from a cross of C. arietinum (ICC-4958) and C. reticulatum
(PI 489777) was used in this study. The RILs were developed by the
single-seed descent procedure. Scoring for resistance to fusarium wilt
in the two parents and 96 RILs was done in the greenhouse. Results

DNA extraction
We previously demonstrated that when the simple se-
quence repeat (AC)8YT is used directly in a PCR reac-
DNA was isolated from vegetative buds and leaf tissues of the parents tion it amplifies a marker, UBC-855500 , which is linked
and RILs using the microprep method of Doyle and Doyle (1987). to the gene for resistance to fusarium wilt race 4 (Rat-
One gram of each sample was submerged in liquid nitrogen and then naparkhe et al. 1988). The aim of our present study was
ground to a fine powder. The powder was quickly transferred to to determine the suitability of the (AC)/ sequences as
a tube containing 7.5 ml of ice cold extraction buffer (0.35 M sorbitol,
0.1 M TRIS, 5 mM EDTA, pH 7.5). The tube was briefly shaken, and guidelines for selecting new primers for use in marker
7.5 ml of nuclei lysis buffer (2 M NaCl, 0.2 M TRIS, 50 mM EDTA, enrichment at the desired region. For this we studied
2% CTAB, pH 7.5) was then quickly added, followed by 3 ml of 5% the inheritance of ISSR polymorphism in a cross of
sarkosyl solution. Sample sets were incubated in a 65°C waterbath for cultivated chickpea (C. arietinum) and the closely re-
20 min. After incubation, the tubes were allowed to cool for a few
minutes and then 18 ml of chloroform/isoamyl alchohol (24 : 1) was
lated wild species (C. reticulatum). Based on the
added to each tube. The tubes were then centrifuged at 500 g for (AC)8YT sequence which amplifies a marker linked to
15 min. The aqueous layer was removed and extracted again with the disease resistance gene, we selected other sequences
15 ml chloroform mixture. Finally DNA was precipitated with chilled containing AC repeats, but ones that varied at the
ethanol and suspended in 1 ml of TE buffer. 3@ and 5@ anchors. We found that other SSRs with AC
repeats also amplified fragments that were linked to the
Culture preparation and inoculation procedures fusarium wilt resistance genes. The repeat (AC)8T am-
plified a marker UBC-8251200 which was located
Inoculum was prepared from a single-spored fungal isolate grown 5.0 cM from the gene for resistance to fusarium wilt
on sterile filter paper placed on potato-dextrose-agar (PDA) as race 4 and was closer than the UBC-855500 and
 517

CS-27700 markers (Mayer et al. 1997; Ratnaparkhe Mayo 1972; Islam et al. 1993) and downy mildew resist-
et al. 1998). The electrophoretic pattern of the PCR- ance gene in lettuce (Hulbert and Michelmore 1985).
amplified DNA fragment using primer UBC-825 is We studied the inheritance and segregation of the gene
shown in Fig. 1. Repeat (AC)8YG amplified fragment for resistance to fusarium wilt race 5, which was found
UBC-857800 which was located 5.5 cM from UBC- to be in the same linkage group with race 4. The genes
855500 and was associated with the gene for resistance for resistance to fusarium wilt race 1, 2, 4 and 5 were
to fusarium wilt trace 4. A one or two nucleotide also found to be clustered in another cross, WR-
change at the 3@ end in the sequence resulted in the 315]C-104 (Tullu 1996).
shifting of the marker. To study the inheritance of the We observed an abundance of dinucleotides and
complimentary sequences, we selected (TG)/ repeats trinucleotide repeats at the fusarium wilt disease resist-
with different anchors. The repeat (TG)8RG amplified ance gene cluster. In addition to (AC)/ and (TG)/ re-
fragment UBC-860600 from the resistant lines, and not peats the trinucleotides (ATG)6, (CTC)6 and (GAA)6
the UBC-857800 of the (AC)/ repeats, and flanked the were also present at the fusarium wilt resistant gene
gene for resistance to race 4. cluster. The markers amplified by trinucleotide repeats,
UBC-864425 , UBC-866900 , and UBC-868700 , were as-
sociated to the genes for resistance to fusarium wilt and
were in the same linkage group. The sequence of the
Genes for resistance to fusarium wilt races 4 and 5 repeats and the size of the amplified markers are shown
are present in same linkage group in Table 1. The position of ISSR markers with respect
to the gene for resistance to race 4 (Foc 4) and 5 (Foc 5)
Recent study indicates that host resistance genes are are shown in Fig. 2. The size of the amplified bands and
often clustered as a multiallelic series at locus or as the position of various markers indicate that they were
multiple linked loci. The best examples are rust resist- amplified from the independent loci and are not the
ance genes clustered in the L group of flax (Sheperd and same fragment. Our study shows that markers linked to
various genes can be rapidly identified using the ISSR-
directed approach. The presence of dinucleotide and

Fig. 2 Position of the genes for

resistance to race 4, 5 and ISSR
markers. The distances among
the markers are given in
centiMorgans

Fig. 1 PCR-amplified inter-simple-sequence-repeat patterns on 2%

agarose gels. M Marker pBR 322 BstN1 digest, lanes 1–12 chickpea
RILs amplified with primer UBC-825. Marker UBC-825 is
1200
5.0 cM from the gene for resistance to fusarium wilt race 4 and is
indicated by an arrow

Table 1 Segregation of ISSR

fragments in chickpea RILs Primer no. Sequence! Fragment s2 P
developed from a cross of ICC- size (bp) (1 : 1)
4958 and PI 489777
UBC-825 ACACACACACACACACT 1200 1.36 0.25—0.50
UBC-855 ACACACACACACACACYT 500 4.0 0.02—0.05
UBC-857 ACACACACACACACACYG 800 3.3 0.05—0.10
UBC-860 TGTGTGTGTGTGTGTGRA 600 4.6 0.02—0.05
UBC-864 ATGATGATGATGATGATG 425 2.0 0.10—0.25
UBC-866 CTCCTCCTCCTCCTCCTC 900 0.52 0.25—0.50
UBC-868 GAAGAAGAAGAAGAAGAA 700 1.3 0.25—0.50

! R indicates purine; Y indicates pyrimidine

518

gene (Rj2). In lettuce (¸actuca sativa L.) 13 resistance

genes for downy mildew (Bremia lactucae Regel) have
been mapped in four clusters (Hulbert and Michelmore
1985). In the present study we found that the genes for
resistance to fusarium wilt races 4 and 5 are linked. The
clustering of resistance genes at a specific chromosomal
region is advantageous in a breeding program, as this
block of genes can be transferred to an adapted back-
ground via backcrossing, and the desired trait can be
selected using the ISSR markers. Our results also in-
dicated the presence of SSRs at the disease resistance
gene cluster and the fragments amplified by SSRs often
co-segregated, indicating a cluster of the microsatellite
repeat. The clustering of microsatellites has also been
reported in tomato where mapping of GATA- and
Fig. 3 Electrophoretic patterns of PCR-amplified inter-simple se- GACA-containing microsatellite loci showed that they
quence repeats on 2% agarose gels. M Marker pBR 322 BstN1 were not randomly distributed throughout the genome
digest, lanes 1–12 chickpea RILs amplified with primer UBC-880 but often clustered in the same chromosomal region
(Arens et al. 1995; Broun and Tanksley 1996). The
remarkably high level of polymorphism around the
tetranucleotide repeats can be exploited for fine map- disease resistance gene cluster indicates an association
ping of fusarium wilt gene cluster. between the molecular mechanism of disease resistance
Our study indicated that markers amplified with and rapid sequence divergence in plants (Sudupak et al.
simple sequence repeats co-segregated and were often 1993; Yu et al. 1996). The multiple genes for disease
present in the same linkage group. The clustering of resistance are thought to be due to the duplication of
markers was noted in several cases. For example, frag- the ancestral gene. It might be possible that the region
ments UBC-8801500 and UBC-8801700 co-segregated surrounding the gene containing SSR repeats was also
at a distance of 5.8 cM and were in the same linkage duplicated and thus provided amplifications using SSR
group (data not shown). The segregation of markers sequences. The shift in the position in the marker varied
amplified by primer UBC-880 is shown in Fig. 3. according to the anchors present at the 3@ end and
Markers UBC-8561800 and UBC-8591900 amplified by 5@ end of the SSR sequence. The anchors at the 5@ end
the complementary repeats (AC)8YA and (TG)8RC, had a small change in the position of the marker. By
respectively, co-segregated and were clustered. changing the sequence of the short nucleotide residues
at the ends, many primers with different anchors can be
synthesized which may amplify markers more closely
linked to the disease resistance gene. Additional experi-
Discussion ments with ‘in situ’ hybridization would provide de-
tailed information on the association of various SSRs
There are many reports of microsatellite markers with disease resistance gene clusters.
linked to disease resistance genes (Yu et al. 1996; Blair
and McCouch 1997). However, finding SSR markers
linked to disease resistance genes is very expensive and Applications of the ISSR-directed approach
involves screening the library, sequencing the clone and
synthesizing the primers. This has prevented the broad In summary, our results indicate that SSRs can be used
use of microsatellites in plants. Our study demon- as highly informative markers for genome mapping and
strated that microsatellite sequences can be used dir- gene tagging. The ISSR-directed approach in combina-
ectly in the PCR reaction and can be used to find tion with bulked segregant analysis (BSA) has a wide
markers linked to disease resistance genes. This ap- application in plant and animal genome mapping. It
proach is quicker and less expensive than southern can be extremely useful in (1) identifying the markers at
hybridization and oligonucleotide fingerprinting. The clusters of disease resistance genes (2) filling large gaps
polymorphism is detected within the region spanned by in linkage maps (3) developing the sequence-tagged
the microsatellite primer and also within the amplified microsatellite sites and (4) providing marker enrich-
region between the primer binding sites. ment at desired regions.
The clustering of host resistance genes conditioning
resistance to pathogenic fungi has been documented in
Acknowledgements Helpful suggestions by Drs. C. A. Ryan, T. W.
many plant species. In soybeans, Lohnes et al. (1993) Okita, V. R. Franceschi, P. K. Ranjekar and V. S. Gupta are
reported that two resistance genes (Rmd and Rps2) are gratefully acknowledged. We also thank Drs. S. S. Jones and C. J.
closely linked to each other, and to a non-nodulation Coyne for critical review of the manuscript and D. Tinnemore for
 519

technical assistance. This work was supported by the McKnight Mayer MS, Tullu A, Simon CJ, Kumar JS, Kraft JM, Kaiser WJ,
Foundation through a collaborative project between Washington Muehlbauer FJ (1997) Development of DNA marker for fusar-
State University, Pullman, Washington, USA and Plant Molecular ium wilt resistance in chickpea. Crop Sci 37 : 1625—1629
Biology Unit, National Chemical Laboratory, Pune, India. Morgante M, Olivieri AM (1993) PCR-amplified microsatellites as
M. B. Ratnaparkhe wishes to thank the McKnight foundation for markers in plant genetics. Plant J 3 : 175—182
providing the Fellowship that made the research possible. Nandal I, Deubelbeiss C, Guttenbach M, Epplan JT, Schmid
M (1990) Heterogeneities in the distribution of (GACA) simple
/
repeats in the karyotypes of primates and mouse. Hum Genet
85 : 187—194
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