80 Rice Genetics Newsletter Vol.23
22. Molecular mapping of resistance to tungro virus in rice cultivars Vikramarya and Utri Rajapan
C. N. NEERAJA, D. KRISHNAVENI, K. SAIVISHNUPRIYA, G. S. V. PRASAD and K.
MURALIDHARAN
Directorate of Rice Research, Rajendranagar, Hyderabad, 500030 India
Sporadic rice tungro virus disease outbreaks in India lead to a maximum production loss of 53% in
a district and 2% in the country. An epidemic during 2001 in three districts of West Bengal caused an
un-milled rice production loss of 0.5 million tons valued at US $ 65 million (Muralidharan et al. 2003a).
Saito et al. (1976) and Hibino et al. (1978) assumed tungro disease to be caused by a unique
combination of a spherical picorna virus (RTSV) and a bacilliform DNA pararetro virus (RTBV). Green
leafhopper (GLH) species Nephotettix virescens is the most dominant vector that transmits virus
particles in India (Siddiq et al. 1994). RTBV has been shown to be integrated with the host genome
(Harper et al. 2002). Based on all the circumstantial evidence, Muralidharan et al. (2003b) showed
tungro to be primarily caused by RTSV. Resistance to RTSV was suspected to be located on chr. 11 in
Oryza officinalis (Kobayashi et al. 1992). In ARC 11554, resistance to both RTSV and GLH appeared to
be tightly linked and controlled by a dominant gene located within 5.5 cM of RFLP marker RZ262 on
chr. 4 (Sebastian et al. 1996). In Utri Rajappan, two dominant and complementary genes were shown to
confer resistance to RTSV (Prasad et al. 2004). RTSV resistance in Utrimerah was reported to be
monogenic recessive and located on chr. 7 near 78cM (~22 Mb) of marker RM336 (Choi et al. 2005).
Two cultivars, Vikramarya (India, IET 7302) and Utri Rajapan (Indonesia, IRG ACC. No. 16684) were
known to possess resistance (score 1) to RTSV (Ebron et al. 1994, Prasad et al. 2004). We studied the
genetic basis of resistance to RTSV in these two cultivars by using QTL analysis. Two mapping
populations were developed using susceptible cultivar TN1 (score 9) x Vikramarya, and TN1 x Utri
Rajapan. The F2 progenies of both mapping populations along with parents were screened for the reaction to RTSV in a glasshouse (28±2°C, >95% RH) using a locally virulent population of N.
virescens. Seeds of each F2 progeny and parents were sown singly in lines in plastic trays (60 x 40 cm) at a spacing of 5 cm between plants and 20 cm between lines. Initially GLH was provided with an
acquisition feeding on RTSV infected plants for 12 h. Fifteen-days old seedlings were individually
capped with a Mylar cage into which 2-3 viruliferous GLH were released for 24 h and the reaction was
scored 15 days later. There was no mortality of insects in any of the inoculations made on using RTSV
carrying GLH after acquisition feeding on infected plants or RTSV-free GLH. The parents TN1
succumbed to RTSV while Utri Rajapan and Vikramarya remained resistant. The RTSV resistance in
both F2 progeny populations showed continuous frequency distribution (Fig. 1).
Research Notes 81
Parental polymorphism was studied using 120 evenly distributed rice microsatellite markers covering the 12 chr. (Chen et al. 1997). In Utri Rajapan and Vikramarya, 58 and 63 markers were found polymorphic. As selective genotyping was shown to be effective to identify specific associated regions of the chromosomes (Nandi et al. 1997), 20 plants each showing extremely resistant and susceptible reactions to tungro were assayed individually with all these markers. Initially, markers RM 542 (chr. 7) and RM6844 (chr. 2) in Utri Rajapan, and RM427 (chr. 7) and RM6902 (chr. 1) in Vikramarya were found to be associated with RTSV resistance.
Fig. 1. Frequency distribution of phenotypic reaction of F2 progeny to tungro spherical virus in individual inoculation test using Nephotettix virescens as vector.
Utri Rajapan Vikramarya
Chr. 7 Chr. 2 Chr. 7 Chr. 1
RM542 RM6844 RM495 RM427
RM13530 RM21576 (22.7 Mb) RM21135 (5.0 Mb) RM10123 (2.3 Mb) (17.2 Mb) RM21640 RM13608 RM10133 (2.6 Mb) (24.7 Mb) RM21205 (6.3 Mb) (18.6 Mb) RM6902 RM320 RM526
Fig. 2. Chromosomal locations of QTLs for tungro spherical virus resistance in two mapping
populations. Black bars indicate marker intervals of QTLs detected.
82 Rice Genetics Newsletter Vol.23
Table 1. Putative QTLs detected for resistance to tungro spherical virus in F2 progeny mapping populations from the crosses between susceptible (S) and resistant (R) cultivars
Cross combination Associated markers Chr. Peak LOD PV % Additive effect TN1/Utri Rajapan RM21576 – RM21640 7 18.0 40.8 1.6 RM13530 – RM13608 2 9.4 21.6 0.9 TN1/Vikramarya RM21135 – RM21205 7 12.2 18.7 0.8 RM10123 – RM10133 1 11.5 16.4 0.6
A 3 Mb region of the sequence encompassing the associated marker was considered and based on
the number of repeats, ~50 microsatellite markers were selected. They were used to survey the parental
polymorphism and to screen all 220 F2 plants. A local linkage map was constructed using Mapmaker Exp 3.0 for each of the four genomic regions and QTLs were identified using Mapmaker QTL version
1.1 (Table 1, Fig. 2). Two QTLs controlling RTSV resistance were detected on chr. 7 and 2 in Utri
Rajapan explaining 40.8 and 21.6 % of the phenotypic variance, respectively, and two on chr. 7 and 1 in
Vikramarya explaining 18.7 and 16.4 %, respectively. From the sequence information (www.tigr.org),
we have deduced the putative candidate genes for resistance in the mapped regions that included NB-
ARC, LRR, protease inhibitors and serine threonine kinases. Further work is in progress to complete the
high-resolution mapping.
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