80 Rice Genetics Newsletter Vol.23

22. Molecular mapping of resistance to tungro virus in rice 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 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 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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Research Notes 83

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