Tropical Plant Biol.
(2010) 3:219–226
DOI 10.1007/s12042-010-9059-9
Flanking Microsatellite Markers for Breeding
Varieties Against Asian Rice Gall Midge
Arundhati Nanda & Sangram Keshori Mohanty &
Rudraksh Sovan Panda & Lambodar Behera &
Anand Prakash & Sarat Chandra Sahu
Received: 2 June 2010 / Accepted: 10 November 2010 / Published online: 14 December 2010
# Springer Science+Business Media, LLC 2010
Abstract The Asian rice gall midge, Orseolia oryzae Wood-
Mason (Cecidomyiidae: Diptera) is a serious pest of wet
season rice in South and Southeast Asia. Due to internal
feeding habit and presence of biotypes of the pest, the most
feasible way to control is breeding varieties resistant against
multiple biotypes through marker-assisted breeding (MAB).
But very few versatile co-dominant markers linked to the gall
midge resistance genes are available. We used a set of F9
recombinant inbred lines (RILs) of the cross TN1/PTB10 and
identified microsatellite markers for the gall midge resistance
gene in cv. PTB10 on short arm of rice chromosome 8.
Markers RM22550 and RM547 flank the gene at a distance
of 0.9 and 1.9 cM, respectively. Amplification of the markers
in gall midge resistant and susceptible cultivars showed that
these markers can be successfully used in MAB for
development of gall midge resistant varieties.
Keywords Biotype2 . Microsatellite markers . Orseolia
oryzae Wood-Mason . Oryza sativa L. . Resistance gene
Introduction
The Asian rice gall midge, Orseolia oryzae Wood-Mason
(Cecidomyiidae: Diptera) is an important pest of the
Communicated by: Hongwei Cai
Electronic supplementary material The online version of this article
(doi:10.1007/s12042-010-9059-9) contains supplementary material,
which is available to authorized users.
A. Nanda : S. K. Mohanty : R. Sovan Panda : L. Behera :
A. Prakash : S. C. Sahu (*)
Pest Genomics Laboratory, Central Rice Research Institute,
Cuttack, Orissa 753 006, India
e-mail: scsahu_crri@yahoo.co.in
irrigated and shallow water rice ecosystems in Asia and
Southeast Asia during the wet season. The maggots feed on
the growing tip, causing the formation of a tubular leaf
sheath, called ‘silver shoot’, in place of inflorescence. The
estimated worldwide loss caused by gall midge alone is
more than US$ 550 millions per year (Herdt 1991).
Chemical control is inefficient due to the internal feeding
habit of the pest and the prevailing hydrological and
edaphological conditions during the wet season. Use of
resistant varieties has been the most feasible alternative to
manage the pest. Ample sources of resistance are available
in cultivated rice, Oryza sativa L. Inheritance studies have
identified nine dominant and one recessive gene imparting
resistance to different biotypes of Asian rice gall midge
(Kumar et al. 2005; Pani and Sahu 2000), and eight of them
have been tagged with different molecular markers. Mohan
et al.(1994) identfied two RFLP markers, RG329 and
RG476, linked to the gall midge resistance gene, Gm2 in
the rice cv. Phalguna. RAPD markers, OPF81700 and
OPF10 600 , and microsatellite markers, RM241 and
RM317, were also identified subsequently for the Gm2
resistance gene (Nair et al. 1995; Sundaram et al. 2003).
Microsatellite markers, RM219, and RM444 were identi-
fied to be linked to gall midge resistance gene, Gm1 in rice
cv. W1263 (Biradar et al. 2004). RAPD markers, E20570
and RFLP markers, R1813 and S1633B were identified
linked to the gall midge resistance gene, Gm4 in rice cv.
Abhaya (Mohan et al. 1997; Nair et al. 1996). RAPD
markers, OPQ12; OPB14 and OPQ05 were identified to be
linked to gall midge resistance genes, gm3 and Gm5,
repectively (Katiyar et al. 2000; Lima et al. 2007). Dubey
and Chandel (2010) conducted in silco survey and localised
Gm4 between markers, RM210 and RM256 spanning a
region of 2.056 Mb on long arm of chromosome 8 and
Gm5 between RM101 and RM309 spanning a region of
220
13.2 Mb covering parts of both the arms of chromosome 12
across centromere. A RAPD marker, OPM61400 and a
RFLP marker, XRG214 were found linked to the gall
midge resisstance gene, Gm6 in the rice cv. Duokang
(Katiyar et al. 2001). AFLP derived SCAR markers, SA598
on chromosome4 was found to be linked to the gall midge
resistance gene, Gm7 in the rice cv. RP2333-156-8 (Sardesai
et al. 2002; Jain et al. 2004) identified an AFLP marker,
AR257 and RAPD marker, AP19587 on chromosome8
linked to the gall midge resistance gene, Gm8 in the
rice cv. Jhitipiti. But these markers were not successfully
used in breeding programs as they were either genotype
specific, distantantly located, or completely dominant.
Microsatellite or simple sequence repeat (SSR) markers
are considered to be the best because they are co-
dominant, multi-allelic, highly polymorphic, reliable, dis-
tributed throughout the genome and can be easily analysed
using polymerase chain reaction (McCouch et al. 1997).
These markers have been extensively used in genome
mapping, gene tagging, evaluation of genetic diversity,
phylogenetic studies, identification and determination of
parentage, purity checking of hybrids, etc. in different crop
species. Many disease and insect pests resistance genes in
rice have been tagged and mapped using microsatellite
markers (Chen et al. 2006; Fujita et al. 2006; Gu et al.
2004; Jena et al. 2006; Sirithunya et al. 2002; Xi-ming et al.
2004)
Tightly linked co-dominant SSR markers for Gm1 and
Gm2 gall midge resistance genes have been developed
(Himabindu et al. 2007). Gm1 is resistant to biotypes 1, 3,
5, and 6 while Gm2 is resistant to biotypes 1, 2, and 5.
Although these two genes impart resistance against multiple
biotypes, both lack resistance against biotype4. PTB10, an
indica land race from Kerala, India, is resistant to
biotypes1, 2, 3, and 4 (Pasalu and Rajamani 1996).
Inheritance studies show that a single dominant gene in
PTB10 imparts resistance against biotypes1, 2 and 4
(Sahu et al. 2004). Srivastava et al. (1994) identified a
single dominant resistance gene, Gm4 in rice cv. Abhaya
(a derivative of PTB10) against biotype1. Closely linked
SCAR (Sequence Characterized Amplified Region)
markers and flanking RFLP (Restriction Fragment Length
Polymorphism) markers for the Gm4 gene have been
developed (Mohan et al. 1997; Nair et al. 1996). RFLP
analysis requires greater laboratories facilities, specialised
equipments, costly and is not suitable for MAS breeding
programs.
The present investigation aimed to identify closely
linked flanking microsatellite markers for the gall midge
resistance gene in PTB10 and assess the potentiality of
these markers for incorporation of biotype4 resistance in
molecular breeding programs for evolving durable gall
midge resistant varieties.
Tropical Plant Biol. (2010) 3:219–226
Methods
Selection of Experimental Materials
The plant materials consisted of susceptible parent TN1,
resistant parent PTB10 and 112 F9 RILs of the cross TN1/
PTB10 developed through single-seed-descent method.
Nineteen rice cultivars resistant/susceptible to gall midge
were used to test the suitability of the markers for MAB.
Biotype2 population of the Asian rice gall midge prevalent
at the experimental farm of Central Rice Research Institute,
Cuttack, India was reared and maintained on susceptible cv
TN1 and used in the studies.
Screening for Gall Midge Resistance
The parents, 112 RILs and nineteen rice cultivars were
screened for resistance/susceptibility to biotype2 under
artificial infestation conditions following Kalode et al.
(1967). Single plant of RILs and rice cultivars were grown
in rows (25 plants in each row) in zinc trays with one row
each of susceptible and resistant parent in each tray. When
plants were 20 days old, male and female gall midges (in
ratio of 1:2) were released per tray in an insect proof cage.
Plant evaluations were considered authentic when all the
TN1 plants had silver shoots and PTB10 had no silver shoot.
Microsatellite Primers and PCR Amplification
Seventy-five rice microstellile loci specific primers distributed
on rice chromosome 8 were used in the study. The primer
sequences were downloaded from Gramene database (http://
www.gramene.org) and custom synthesized by Qiagen
Operon Technologies, Almeda, California, USA. Genome
scanning for microsatellite polymorphism was done in two
steps: 47 microsatellite specific primers were used to
determine the chromosomal location of the gene in PTB10
and then 28 additional microsatellites specific primers to
identify closely linked flanking markers for the gene.
Genomic DNA from parents, individual RILs and nine-
teen cultivars were isolated according to the methods of
Murray and Thompson (1980). Equal amount of genomic
DNA from each of 15 homozygous resistant (R) and 15
homozygous susceptible (S) RILs were pooled to form
resistant and susceptible bulks, respectively. The concen-
tration of the two bulks and two parental DNAs was
adjusted to 15 ng/μl.
Bulk segregation analysis (BSA) was done following
Michelmore et al. (1991) with slight modifications. Ampli-
fication of genomic DNA of parents and pooled genomic
DNAs of homozygous R and S lines was carried out in a
thermal cycler (Perkin Elmer, Cetus, USA). The 20 μl
reaction mixture contained 30 ng of genomic DNA, 1X
Tropical Plant Biol. (2010) 3:219–226 221

PCR buffer {75 mM Tris–HCl (pH 9.0), 50 mM KCl,
20 mM (NH4)2SO4}, 200 μM dNTP mix (MBI Fermantas,
Lithuania, USA), 4 picomole of each of forward and
reverse primers, 2 mM of MgCl2 and 1U of Taq DNA
polymerase (Biotools, Spain), overlaid with one drop of
mineral oil. Thermal profile for amplification was; initial
denaturation temperature of 93°C for 3 min followed by 36
cycles of denaturation temperature of 93°C for 1 min,
annealing at 55–67°C (depending upon Tm value of
primers) for 1 min and extension at 72°C for 1.5 min and
final extension at 72°C for 5 min. The amplified products
were size fractionated on a 2.5% agarose gel containing
ethidium bromide and visualized under UV using Gel
Imaging System (Fluor ChemTM 5500, Alpha Innotech,
USA). The size of amplified fragments was determined
using Alphaease software (Alpha Innotech, USA).
marker order was determined using a LOD score of 3.0 and
above, and recombination frequency was converted to map
distances by Kosambi function (Kosambi 1944).
Results
Evaluation of Gall Midge Resistance
The resistant parent, PTB10 showed no silver shoot and the
susceptible parent, TN1, showed 100% silver shoots. Thus
insect pressure was sufficient for evaluating the plants.
Thirty two RILs were homozygous resistant, seventy eight
were homozygous susceptible, and two showed residual
heterozygosity. Out of the 19 rice cultivars, 9 were
susceptible and 10 were resistant to biotype2 (Table 1)

Linkage Analysis Identification of Flanking Microsatellite Markers

The linkage of marker was determined using 110 RILs and Out of 47 SSR primers, 37 primers amplified polymorphic
MAPMAKER software v3.0 (Lander et al. 1987). The loci between TN1 and PTB10. Five microsatellite primers,

Table 1 Reaction of rice cultivars to biotype2 of rice gall midge and allelic diversity of markers flanking the resistance gene

Sl. No. Rice cultivars Parentage Gall midge resistance Reaction to gall Marker allele
gene(s) midge biotype2 amplification

RM547 RM22555 RM22550

1 IR64 IR5657/IR2061 – S P T P
2 Lalat Vikram/W1263 Gm1 S P P P
3 Leung 152 Land race from Thailand Gm2 R P, T P, T P, T
4 NHTA8 Land race Gm1+ Gm9(t), Gm 10t) R T T T
5 Siam29 Land race from Thailand Gm2 R P P P
6 Vellutha- Land race gm3 R P P P
cheera
7 W1263 MTU15/Eswarakora Gm1 S OA T P, T
8 Samridhi IR22/W1263 Gm1 S P P P
9 ARC 5984 Land race Gm5 R OA T OA
10 Banglei Land race Gm3(t), Gm5(t), Gm 11(t) R T T T
11 Surekha IR8/Siam29 Gm2 R T T T
12 PTB33 Pure line selection from – S OA T T
Arikarai
13 Kavya Mahsuri/Surekha Gm1 S T T T
14 Swarna Vasishta/Mahsuri None S T T T
15 TN1 DGWG/TSAIYUANCHUNG None S T T T
16 Phalguna IR8/Siam29 Gm2 R T T T
17 Abhaya CR157-392/OR57-21 Gm4 R P P P
18 PTB10 Breeding line from Patambi Gm4 R P P P
19 Samba GEB24/TN1/Mahsuri – S – T OA
Mahsuri

P: Marker allele linked to resistance

T: Marker allele linked to susceptibility
OA: Other allele
222
RM152, RM547, RM3215, RM6208 and RM6429 ampli-
fied polymorphic loci between parents, and bulked R and S
DNA (Supplementary Table 1). Co-segregation analysis
with individual RILs constituting the pooled DNA showed
that the marker RM547 amplified a locus of 225 bp in the
resistant parent and in all R lines, and heterozygous loci of
203 bp and 298 bp in the susceptible parent and all S lines
(Fig. 1a, b). Primer, RM152 amplified fragment of 150 bp
in nine R lines, and one S line while heterozygous
fragments of 143 bp and 170 bp in 14 S lines and 6 R
lines (Fig. 1c, d). RM6208 amplified fragments of 143 bp
in fourteen R lines, and 149 bp in 15 S lines and one R line.
RM 6249 amplified fragments of 100 bp in 14 R lines, and
106 bp in 15 S lines and one R line. RM3215 amplified
fragments of 90 bp in 11 R lines, and 200 bp in 15 S lines
and 4 R lines (Supplementary Table 2).
Using primers, RM152, RM547, RM3215, RM6208 and
RM6429, and 110 RILs, the linkage and position of the
gene relative to these markers was estimated. The order
observed was RM152-gene-RM547-RM6208-RM6429-
RM3215. This was consistent with the rice genetic map of
the same SSRs on the short arm of chromosome 8 (www.
gramene.org). The genetic distances between the gene and
these markers were 14.6 cM, 1.9 cM, 2.8 cM, 4.8 cM, and
11.2 cM, respectively (Table 2).
Subsequent analysis with 28 microsatellite primers
around RM547 and in a physical distance of 4.78 Mb to
5.98 Mb, showed seven primers RM22506, RM22551,
RM22550, RM22555, RM22563, RM22564 and RM22565
amplified polymorphic loci between parents and bulked R
and S DNAs. Co-segregation analysis with individual R
and S lines showed that RM22506, RM22550, RM22563,
RM22564 and RM 22565 amplified fragments of 350 bp,
260 bp, 319 bp, 397 bp, 395 bp and 225 bp in all R lines
and fragments of 460 bp, 285 bp, 335 bp, 380 bp, 380 bp,
and 200 bp in all S lines, respectively. RM22551 amplified
heterozygous loci of 143 bp and 202 bp in all 15 R lines
Fig. 1 Co-segregat ion of
RM547225 and RM152160 a b
markers with resistance (a & c, Resistant RILs
respectively) and RM547
203&298 and RM152 143&170 with
susceptibility (b & d, respec-
tively) to gall midge. Rp–resis-
tant parent, Sp–susceptible
parent, M -molecular weight
markers (50 bp DNA ladder), c d
arrows on the left margin indi- Resistant RILs
cate the resistant/susceptible
* * * * *
specific markers, numbers on
the right margin represent mo-
lecular weight markers in bp,
* indicates the recombinant RIL
Tropical Plant Biol. (2010) 3:219–226
and one S line and heterozygous loci of 135 bp and 180 bp
in 14 S lines (Supplementary Table 2). Fig. 2a, b, c and d
show the amplification of fragments by closely linked
flanking microsatellite loci, RM22550 and RM22555,
respectively from resistant and susceptible RILs constitut-
ing bulks.
Using primers RM22506, RM22551, RM22550,
RM22555, RM22563, RM22564 and RM 22565 and all
110 RILs, the linkage and position of gene relative to these
markers was estimated. The order observed was RM22506-
RM22551-RM22550-gene-RM22555-RM22563-
RM22564-RM22565. This was consistent with the rice
genetic map of the same SSRs on the short arm of
chromosome 8 (www.gramene.org). The genetic distances
between the gene and these markers were 5.8 cM, 1.9 cM,
0.9 cM, 1.9 cM, 2.8 cM, 2.8 cM and 2.8 cM, respectively
(Table 2).
The linkage and position of the gene relative to the 12
markers was mapped (Fig. 3). The order observed was
RM152-RM22506-RM22551-RM22550-gene-RM547-
RM22555-RM6208-RM22563-RM22564-RM22565-
RM6429-RM3215. The order was consistent with the rice
genetic map of the same SSRs on short arm of rice
chromosome8 (www.gramene.org). RM547 and RM22555
are located at 1.9 cM from the gene. Physically RM547 is
in proximity to the gene than RM22555.
To evaluate the utility of the flanking markers in marker-
assisted- breeding programs, amplification of the marker
alleles in 19 land races and elite cultivars that differed in their
reaction to rice gall midge biotype2 was done. Marker,
RM547 amplified 225 bp resistance-linked allele (P allele) in
IR64, Lalat, Leung152, Siam29, Velluthacheera, Samridhi,
Abhaya, and PTB10 and susceptible-linked heterozygous
203 bp and 298 bp fragments (T allele) in Leung152, NHTA8,
Banglei, Surekha, Kavya, Swarna, TN1 and Phalguna
(Table 1). Other alleles (OA allele) of 215 bp, 250 bp and
265 bp were amplified in PTB33, W1263, and ARC5984,
RpSpM Susceptible RILs RpSpM
500
300
200
100
RpSpM Susceptible RILs RpSpM
* *
500
300
200
100
Tropical Plant Biol. (2010) 3:219–226 223

Table 2 Co-segregation of resistance with microsatellite markers

Sl. No. Markers Allele No of resistant No of susceptible No of No of non- Map distances from resistance
RILs RILs recombinants recombinants gene (in cM)
P/Ta bp

1 RM152 P 150 23 5 14 96 14.6

T 143/170 9 73
2 RM547 P 225 31 1 2 108 1.9
T 203/298 1 77
3 RM6208 P 143 31 2 3 107 2.8
T 149 1 76
4 RM6429 P 100 28 1 5 105 4.8
T 106 4 77
5 RM3215 P 190 22 1 11 99 11.2
T 200 10 77
6 RM22506 P 350 31 5 6 108 5.8
T 460 1 73
7 RM22550 P 260 31 0 1 109 0.9
T 285 1 78
8 RM22551 P 143/202 31 1 2 108 1.9
T 135/180 1 77
9 RM22555 P 319 31 1 2 108 1.9
T 335 1 77
10 RM22563 P 397 31 2 3 108 2.8
T 380 1 76
11 RM22564 P 395 31 2 3 108 2.8
T 380 1 76
12 RM22565 P 225 31 2 3 107 2.8
T 200 1 76

P: Marker allele linked to résistance

T: Marker allele linked to susceptibility

respectively (Supplementary Fig. 1a). Marker, RM22555
amplified a resistance-linked 319 bp fragment (P allele) in
Lalat, Leung152, Siam29, Velluthacheera, Samridhi, Abhaya
and PTB10 while a susceptible-linked 335 bp fragment (T
allele) in IR64, Leung152, NHTA8, W1263, ARC5984,
Banglei, Surekha, PTB33, Kavya, Swarna, TN1, Phalguna
and Samba Mahsuri (Supplementary Fig. 1b). Marker,
RM22550 amplified a resistance-linked 260 bp fragment (P
allele) in IR64, Lalat, Leung152, Siam29, Velluthacheera,
W1263, Samridhi, Abhaya and PTB10 while a susceptible-
linked 285 bp fragment (T allele) in Leung152, NHTA8,
W1263, Banglei, Surekha, PTB33, Kavya, Swarna, TN1 and

a b
Resistant RILs RPSPM1 Susceptible RILs RpSpM2

500 500
300 300
200 200

c d
Resistant RILs RpSpM1 Susceptible RILs RpSpM2

500 500
300
300

Fig. 2 Co-segregation of RM22550260 and RM22555319 markers with markers (100 bp/50 bp DNA ladder), arrows on the left margin
resistance (a & c, respectively) and RM22550285 and RM22555335 indicate the resistant/susceptible specific markers, numbers on the
markers with susceptibility (b & d, respectively) to gall midge. Rp– right margin represent molecular weight markers in bp
resistant parent, Sp–susceptible parent, M1/M2 -molecular weight
224 Tropical Plant Biol. (2010) 3:219–226

Marker practices like early planting to avoid peak pest incidence at

maximum vegetative stage, less use of nitrogenous fertilizers
cM cM Mb and wider spacing. The resistant variety grown continuously
14.6 RM152 0.683 in a locality becomes susceptible. This may be due to a change
in the virulence of the pest or gradual build-up of a virulent
population, which occurs concomitantly in negligible propor-
tion at the time of deployment of resistance genes. The
effective management strategy is development of varieties
with multiple non-allelic resistance genes to safeguard against
possible change in virulence of the pest or keep the negligible
RM22506 4.725
virulent population at bay. Conventional breeding procedure
5.8 RM22551 5.452 has inherent difficulties for simultaneous incorporation of
RM22550 5.451 many genes. It is only possible through marker-assisted
1.9 R544 (40.2) 5.423 breeding. Eight of the ten gall midge resistance genes in rice
0.9
have been tagged and mapped using different DNA marker
RM547 5.591
1.9 RM22555 5.602
techniques. But their benefit in molecular breeding programs
is yet to be realised because the markers were either genotype
2.8 RM6208 (42.9) 5.783
RM22563 5.796 specific or dominant in nature or not very tightly linked. Co-
RM22564 5.796 dominant SSR markers are most suitable in marker-assisted
RM22565 5.796
breeding and have already been developed for Gm1 and Gm2
R1813 (45.8) (Himabindu et al. 2007) and their success in breeding
programs has been demonstrated. But both the genes lack
S1633B(48.1)
4.8 C1251S (48.8) 8.155 resistance to rice gall midge biotype4. Microsatellite markers
RM6429 (48.80) 8.384 for biotype4 resistance gene will be useful for MAS for
AR257
pyramiding non-allelic resistance for durability.
RM3215 (49.5) 8.567 A single dominant gene in PTB10, an indica land race,
11.2
imparts resistance against biotypes 1, 2, 3 and 4. We
R80, AS168 (50.8) developed F9 recombinant inbred lines of the cross TN1/
Centromere S10715S (50.8) 8.7945
PTB10 and used this population to identify microsatellite
flanking markers for gall midge resistant gene in PTB10.
Using the 75 microsatellite markers, we found that the gall
Fig. 3 Linkage map of short arm of rice chromosome 8 showing midge resistance gene in PTB10 is located within a genetic
positions of molecular markers linked to gall midge resistance genes
distance of 40.2 cM to 42.9 cM and physical distance of
in the present and previous studies. Numbers on the left side of map
shows genetic distances in centimorgans (cM) of linked markers from 5.451 Mb to 5.591 Mb on short arm of chromosome8, and
gall midge resistance gene present in cv. PTB10 obtained in the flanked by RM22550 and RM547. Abhaya, a derivative of
present study while numbers on the right side of map shows physical PTB10 possesses a single dominant gene designated as
distances in Mb and genetic distances in centimorgans (cM)(given in
Gm4 (Srivastava et al. 1994). Nair et al. (1996) identified a
parentheses) on chromosome8, obtained from Gramene database
(www.gramene.org). Markers in italic obtained from Gramene RAPD marker, E20570, linked to Gm4 gene in Abhaya.
database. Genetic and physical distances are not to the scale. shows Mohan et al. (1997) converted the RAPD marker to SCAR
the position of gall midge resistance gene present in cv. PTB10, marker and mapped the marker to short arm of chromosome
identified in the present study. shows the position of gall midge
8 within genetic distance of 45.4 cM to 46.2 cM and
resistance gene, Gm4 present in cv. Abhaya (Mohan et al. 1997; Nair
et al. 1996). shows the position of gall midge resistance gene, Gm8 physical distance of 7.596 Mb to 8.155 Mb between RFLP
present in cv. Jhitipiti (Jain et al. 2004) markers, R1813 and S1633B (Fig. 3). Dubey and Chandel
(2010) conducted in silico survey of resistance gene
Phalguna. Two other alleles (OA allele) of 320 bp and analogues in the genomic region encompassing Gm4 gene
400 bp were also amplified by RM22550 in Samba Mahsuri and suggested that Gm4 gene is located in the long arm of
and ARC5984, respectively (Supplementary Fig. 1c). chromosome 8 between 22.472 Mb and 24.276 Mb. The
different position of gall midge resistance gene in PTB10
and Gm4 in rice cv. Abhaya leads to a suggestion that they
Discussion are different genes although Abhaya is a derivative of
PTB10. These genes may be duplicates integrated in the
The Asian rice gall midge has continued to be a threat to rice different part of the genome but having the same phenotype
agriculture despite the use of resistant varieties, cultural expression. Further studies are needed to confirm the nature
Tropical Plant Biol. (2010) 3:219–226
of gall midge resistance in rice cvs. Abhaya and PTB10. The
flanking microsatellite markers identified in this study offers
the best alternative for MAS breeding programs since it is
PCR based.
Polymorphism of the flanking markers was studied in 19
gall midge resistant and susceptible cultivars for their
suitability in MAS. Resistance-linked marker alleles ampli-
fied by RM22550, RM547 and RM22555 are present in cv.
Lalat, Siam29, Velluthacheera and Samridhi carrying Gm1,
Gm2, gm3, and Gm2, respectively. The resistance specific
RM22550 and RM547 alleles are not amplified in cv.
Phalguna, carrying Gm2 gene. The markers, RM547,
RM22550 and RM22555 amplify different alleles. But
these markers amplify polymorphic loci between PTB10
and Phalguna, Surehka, Kavya, Swarna, and Samba
Mahsuri and thus can be used for successful transfer of
PTB10 gall midge resistance gene into elite susceptible
cultivars like Swarna and Samba Mahsuri and pyramid
Gm1 (from Kavya), Gm2 (from Phalguna) and biotype4
resistance gene (from PTB10) by marker-assisted-breeding
for development of durable gall midge resistant varieties
against biotypes1, 2, 3, 4, 5 and 6.
Acknowledgments The authors gratefully acknowledge the help
and financial support provided by the Director, Central Rice Research
Institute, Cuttack and ICAR, New Delhi, India during the inves-
tigations.
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