Indian Journal of Experimental Biology Vol. 41, April 2003, pp. 346-351

Generation and screening of recombinant inbred lines of rice for yellow stemborer resistance

S Mohankumar*, V Thiruvengadam, K Samiayyan' & P Shanmugasundaram

Centre for Plant Molecular Biology, Tamil Nadu Agricultural University, Coimbatore 641 003, India

'Soil and Water Management Research Institute, Thanjavur 613 501, India

Received 8 August 2002; revised 18 February 2003

Based on the results of studies on varietal screening, antixenosis (egg laying preference) and antibiosis (larval survival and adult emergence); rice varieties W 1263 and C043 were selected as resistant and susceptible parents, respectively, for yellow stemborer (YSB) infestation. A mapping population was developed using above parents following single seed de­scent method. Screening for YSB reaction in FI and F2, generations under field and glasshouse conditions for both dead hearts and white ears, established the polygenic nature of inheritance for YSB resistance. Field screening for YSB resi stance at F9 generation revealed the difference in the reactions among recombinant inbred lines (RILs) between vegetative and re­productive stages. The experiments under field and glasshouse screening of RILs for dead hearts showed significant positive association. However, the reaction was more towards susceptibility in glasshouse screening due to no choice test. Scoring of 250 RILs (Fg) for various morphological traits showed wide range of variation indicating the suitability for QTL mapping.

Two available options for increasing crop production are productivity enhancement and minimizing yield losses caused by biotic and abiotic stresses. Annual losses due to insect pests have been estimated to the tune of $90.5 billion for eight principle crops includ­ing rice'. In rice, the potential losses could touch nearly 55 per cent of expected yield. Among 20 major economic pests of rice, stem borers are the ones that cause severe yield losses. Of the 16 different stembor­ers attacking rice, yellow stemborer (YSB), Scirpo­phaga incertulas Walker causes widespread damage by producing tiller loss (dead hearts) and empty pani­cles (white ears) and causes a steady annual yield loss of 10 million tonnes2

• YSB infestation is often encoun­tered in irrigated lowland, rainfed lowland and deep water, flood-prone rice ecosystems. So, integrated management of YSB is necessary to reduce the crop losses in different ecosystems.

Studies on host plant resistance to YSB have shown that all the three properties of plant defense, viz., antixenosis, antibiosis and tolerance in different proportions contribute to elicit resistant reactions3

-5

.

But lines resistant to stemborer are limited in Oryza sativa. Through conventional breeding, scientists have developed rice varieties with high yield potential and moderate resistance to stemborers. However, the level of resistance is inadequate to meet the challenge of

*Correspondent author- Phone: 091-0422-2457228; Fax: 091-0422-2431672; E-Mail: smk65@hotmail.com

YSB under field conditions. Resistance to YSB dur­ing vegetative and reproductive stages appears to be independent5

. So a durable resistance operative at vari­ous stages of crop growth is essential for management of this pest. A better understanding of the mechanisms and components of resistance, and the genes involved would allow development of rice cuitivars with better and stable resistance to YSB. The present study was carried out to phenotype the recombinant inbred lines with respect to YSB resistance under both glasshouse and field conditions for identifying QTL based on molecular marker approach.

Materials and Methods Selection of parents - To select the resistant and

the susceptible parents for hybridization, reaction of the rice varieties viz., W1263, C043, ASD7, C032, C025, ASDI2, GEB24, ASDIO, ASDI3 , TKM2 and SR-26-B along with the standard checks, TKM6 (resistant) and TN 1 (susceptible) was tested under glasshouse conditions by artificial infestation with YSB larvae. For this, larvae were released on test entries at 14 days after transplanting (DA T) for evaluation as dead hearts (30 DA T) and at boot leaf stage for evaluation as white ears at harvest. The per­centage of dead hearts for each entry was computed using the following formula.

No.of dead hearts Per cent of dead hearts = x 100

Total no. of tillers observed

MOHAN KUMAR et at.: YELLOW STEMBORER RESISTANCE IN RICE 347

Percentage of dead hearts was converted to D which was corrected for the level of infestation. The corrected damage D values were worked out using the formula and the rating scale (0-9) was fixed based on D values as suggested by Heinrichs et al 6

.

D = Per cent dead hearts in test entry x 100

Per cent dead hearts in susceptible check

A separate experiment on antixenosis was con­ducted with these varieties by evaluating the oviposi­tional preference. Young seedlings (25 days old) were transplanted to individual pots and caged separately in a glasshouse. At twenty days after transplanting, ten female moths were allowed to oviposit on each test plant. After four days, the number of egg masses laid by the moths was counted by recording observations on full, half and less than half egg masses in two rep­lications. The identified resistant (W 1263) and sus­ceptible (C043) varieties were re-evaluated along with the standard checks, TKM6 (resistant) and TNI (susceptible) for antibiosis phenomena using the pro­cedure developed at IRRI6. Briefly, a total of 100 first instar larvae were released in twenty plants for each variety. Five replications were maintained. Per cent larval survival and per cent adult emergence were recorded.

Generating mapping population - The resistant (WI263) and the susceptible (C043) varieties were used for generating mapping population. The scheme followed to develop recombinant inbred lines (RILs)

Kh arif 1996

Kharif 1997

Summer 1998

Kharif 1998

Summer 1999 to Summer 200 I

Kharif200 1

Raising susceptible (C043) and resi stant (W 1263) parents and effecting manual crossing

Raising F I hybrids and selfing the true hybrids identified using morphological and molecular markers

Raising F, generation and selfing 250 individuals representing wide spectrum of variability

Rai si ng F, generation (250 single seed descents)

Forwarded to F, generation following si ngle seed descent meth od

Raising F, and phenotypi ng

Fig. I-Schematic diagram showing development of mapping population

is depicted in Fig. 1. Evaluation of F8 RILs for morphological traits­

A total of 250 RILs of F8 generation developed from the cross between C043 and W1263 were used to study the variation in morphological traits. Nine plants were raised for each RIL in a single row by adopting a spacing of 30 cm between rows and 20 cm between plants. Observations were recorded on three randomly selected plants per RIL. Mean, range, vari­ance and SE were worked out for plant height (cm), number of productive tillers per plant, panicle length (cm), number of filled grains per panicle and grain yield (g) per plant. Correlations between YSB damage at vegetative stage and morphological traits, namely, number of internodes, length of 4th internode (cm), stem diameter (mm) and total number of tillers were calculated.

Glasshouse screening of Fl. F2 and recombinant inbred lines-Individual plants of parents, F" F2 and F9 were raised and evaluated independently during Kharif 1997 to Kharif 2001 as detailed in Fig. 1. Single seedling (23 days old) was transplanted in the mud pot. Two weeks after transplanting, the plants were infested with newly emerged larvae (two larvae per tiller) by putting the larvae near the auricle of the young leaf. Caging (putting each pot in a Mylar film cage) was done to avoid escape or movement of lar­vae from one plant to another. Fifteen days after the release of larvae, the plants were evaluated for dead hearts along with susceptible and resistant check. Evaluation was done when 80 per cent of the tillers of susceptible check showed insect infestation.

Field screening of RILs-Two hundred and fifty RILs of F9 generation of the cross between C043 and W 1263 were used for screening. The screening was carried out at the stemborer 'hot spot' area (Soil and Water Management Research Institute (SWMRI), Thanjavur, Tamil Nadu). Two rows (3.5 m) of each RIL were maintained. About 20 plants per RIL were used for scoring of dead hearts and white ears. Dam­age caused was recorded and rated as suggested by Heinrichs et a1 6

. Briefly, the damage was recorded at 50 DA T (when susceptible check showed a minimum of 30 per cent damage) for the dead heart stage and at 5 days before harvest (when susceptible check showed a minimum of 15 per cent infestation) for white ear stage.

Results and Discussion Major difficulties encountered in breeding for YSB

resistance in rice are that varieties resistant to YSB are limited and resistance trait is governed by many

348 INDIAN J EXP 8IOL, APRIL 2003

genes7• Besides, screening for resistance to YSB is

labour intensive, time consuming, and weather/season dependent. Two approaches currently being pursued to breed YSB - resistant varieties are pyramiding genes for resistance using molecular tags and devel­opment of transgenics. Although transgenic lines with Bacillus thuringiensis (Bt) genes8

.9 show promise

against YSB, there is concern about the durability of Bt-based insect resistance 10. Identification of QTLs for YSB resistance, marker-assisted selection (MAS) and gene pyramiding may provide durable resistance and complement conventional breeding programs II. MAS can increase the efficiency and accuracy of selection, especially for traits such as YSB resistance that are difficult to phenotype. The quantitative inheritance of plant resistance to insects is being studied using mo­lecular markers in many cropsl2. With reference to rice pests, QTL mapping has been done for resistance to brown planthopperl3 and white backed planthop­perl4. In the present study, development of mapping population and phenotyping for YSB was done and marker analysis is in progress at Tamil Nadu Agricul­tural University, Coimbatore.

Table 1-Reaction of rice cultivars to yellow stemborer (YS8)

[Values are mean of two replications]

Dead heart stage White ear stage Varieties D value Rating scale D value Rating scale

WI263 17.2 I (R) 6.8 I (R) TKM6 29.8 3 (R) 8.7 I (R) C043 87.6 9 (S) 54.4 7 (S) TNI 74.8 7 (S) 59.2 7 (S) ASD7 78.7 7 (S) 60.4 9 (S) C032 81.9 9 (S) 64.6 9 (S) C025 70.3 7 (S) 51.2 7 (S) ASDI2 80.5 9 (S) 50.2 7 (S) GE824 68.2 7 (S) 54.8 7 (S) ASDIO 80.6 9 (S) 49.2 5 (S) ASDI3 62.3 7 (S) 48.6 5 (S) TKM2 60.7 7 (S) 50.8 7 (S) SR-26-8 68.2 7 (S) 54.2 7 (S)

R: Resistant; S: Susceptible

Screening of thirteen rice vanetles revealed that none of the cultivars was free from damage at both dead heart and white ear stages. The relative damage value D varied from 17.2 to 87.6 in dead heart stage as compared to 6.8 to 64.6 in white ear stage (Table 1). Among the entries, W1263 and TKM6 showed significantly less damage at both the stages. Wl263 was more resistant than TKM6 and assigned scale 1 at both the stages. Study of ovipositional pattern of YSB showed that susceptible cultivars are preferred to resistant cultivars for egg laying. Significantly higher number of eggs was observed on susceptible varieties like TNl, C043 etc. Conversely, varieties W1263 and TKM6, that were rated as resistant were found to have less number of egg masses (Table 2). Thus antixenosis appeared to contribute to YSB resistance. Varieties representing the extreme classes (W 1263 and TKM6 as resistant cultivars and C043 and TN 1 as susceptible cultivars) were evaluated for antibiosis. Larval survival and adult emergence were adversely affected on resistant cultivars Wl263 and TKM6 (Table 3). The slow growth of larvae in resistant cultivars appeared to lead to low degree of damage at

Table 2 - Ovipositional pattern of YS8 in different rice cultivars under glasshouse conditions

[Values are mean of two replications]

Number of egg mass laid by 10 females Varieties Full egg Half egg Less than half

mass mass egg mass

Wl263 0.9 2.1 1.8 TKM6 J.7 2.9 2.4 C043 8.7 12.7 1.4 TNI 7.2 10.4 8.7 ASD7 8.2 4.2 3.1 C032 7.9 8.1 2.6 C025 7.5 5.2 3.8 ASDI2 6.8 4.0 5.4 GE824 5.8 6.4 3.2 ASDIO 8.2 6.8 4.1 ASDI3 8.7 7.2 3.7 TKM2 7.2 4.2 4.8 SR-26-8 5.2 4.8 5.6

Table 3 - Effect of se lected rice cultivars on larval survival and adult emergence of YSB

Dead hearts (%) White ears Larval Adults Varieties

7DI 14 DI 28 DI at harvest survival emerged/100

(%) (%) larvae released WI263

1.4 _ 3.4- 5.7- 2.4- 12.8- 7-

TKM6 3.8b 7.2b lO.4b 5.4b 20.7b 12b

C043 24.Sd 29.8d 32.8c 19.7d 78.4d 48d

TNI 20.l c 23.4c 28 .6c 14.8c 70.6c 36c

DI : Days after infestation Column bearing the same letter are not significantly different at P:s;O.OS level by Duncan's Multiple Ranging Test

MOHANKUMAR et al .: YELLOW STEM BORER RESISTANCE IN RICE 349

dead heart stage. W1263 displayed significantly higher antibiosis than TKM6. Based on the these evaluations which is also supported by the earlier reports4

•7

, W1263 was selected as the resistant parent to cross with the susceptible parent C043 as female to develop mapping population and to study inheritance of YSB resistance.

Most of the Fl plants showed susceptible reaction to YSB with the damage rating of 5-7 as against the ratings of 1 and 9 for the resistant and the susceptible parents, respectively (Fig. 2a). The screening of F2 plants at dead heart and white ear stages revealed wide variation for reaction to YSB. The frequency distribution showed skewness towards susceptibility at both the stages. Some plants, however, were found to be transgressive segregants and were assigned 0 gr~de scale (resistant parent grade scale I) and 9 grade scale (susceptible parent grade scale 9 in dead heart and 7 in white ear stage) (Fig. 2b, c). The F2 dis­tribution of YSB reaction did not fit to any simple Mendelian ratio, but showed continuous variation in­dicating the polygenic nature of resistance. The re­sults suggested that resistance is conferred by reces­sive alleles which is in agreement with earlier findings4

•7

.

A total of 250 RILs (Fg) were scored for various morphological traits. Wide range of variation was ob­served for different traits (Table 4) indicating exten­sive recombination over generations making it an ideal population for mapping the genes of interest. Previous studies l5-16 have found that morphological features of the host plant can influence YSB reaction. In the present study, correlation of morphological characters of RILs with YSB damage revealed stem diameter and total number of tillers do not play sig­nificant role in conferring resistance to YSB whereas, both number of internodes and length of 4th internode from the base were found to be correlated with level of damage in vegetative stage (Table 5).

RILs of F9 generation were used for screening un­der field conditions in hot spot area (SWMRI, Than­javur) and the same population in glasshouse under no choice condition at dead heart stage. Under field con­ditions, there was a significant difference among RILs for YSB resistance between dead heart and white ear stages (Fig. 3a, b). Transgressive segregants were ob­served at white ear stage in both extremes, as com­pared with parents (Fig. 3b). There was a continuous variation in the reaction, which further confirmed the polygenic nature of resistance. The same population

Table 4- Variation for morphological traits among RILs in mapping population in Fs generation

Parameters C043 WI263

RILs Mean Range Variance SE

Plant height (cm) 97 120.7 110.8 75-161 424.36 4.53 Number of productive tillers/plant 17.7 19.7 15.1 6-31 14.52 3.17

Panicle length (cm) 25.5 22.7 22.7 17.7-30.6 4.41 1.24

Number of filled grains/panicle 167 67 .7 117.8 58-177 399.6 15.59

Grain yield(g)/plant 36.6 35.9 24.1 10.7-39.0 41.09 5.75

25 100 30 a 90 b c

20 80

to 70 20

C 15 60 (\l

C. 50 '0

40 ci 10 Z 30 10

5

0 3 5 7 3 5 7 9 3 5 7 9

Damage rating scale

Fig. 2-Frequency distribution of YSB reaction in FI and F2 generations of the cross C0431W1263. [(a) FI-Dead hearts; (b) Fz-Dead hearts; and (c) F2-White ears]

350 INDIAN 1 EXP BIOL, APRIL 2003

80

70

60

50

40

30

20

10

0 0

70

60

50 (/)

...J 40 a: '0

30 ci z 20

10

0 0

70

60

50

40

30

20

PI

P

PI

3 5 7 9

b

3 5 7 9

c

2 3 4 5 6 7 8 9 10

Damage rating scale

Fig. 3 - Transgressive segregation of YSB reaction in RILs at both field and glasshouse conditions [(a) Field-Dead hearts; (b) White ears; and (c) Glasshouse-Dead hearts)

was screened in glasshouse under no choice condition at dead heart stage (Fig. 3c). There was major discre­pancy in reaction to YSB between field and glass­house screenings (Fig. 3a, c) with the reaction more towards susceptibility in glasshouse screening. This may be explained by antixenosis. The parents have shown significant difference for antixenosis (Table 2) and it is expected that RILs would display variation for this trait. Antixenosis would be operative under fie ld screening and thus contribute to YSB resistance,

Table 5 - Correlation of plant morphological traits with yellow stemborer damage in vegetative stage

Parameters

Number of internodes Length of 4lh internode Stem diameter Total number of tillers/plant

** P=O.OI

' r' value (n=246)

0.24** 0.34**

-0. 11 -0.07

whereas in forcefeeding, no choice situation of glass­house, antixenosis component of resistance will not be effective. Hence more number of RILs would be susceptible to YSB. These results also point a way to map loci contributing to antixenosis.

The present study thus demonstrates that YSB re­sistance is under polygenic control and components such as antixenosis, antibiosis and other defense mechanisms contribute to resistance. With the accom­plishment of two major tasks, namely, the develop­ment of mapping population and phenotyping of RILs for YSB resistance, identification of molecular mark­ers linked to YSB resistance is expected to follow soon.

Acknowledgement The authors would like to thank The Rockefeller

Foundation, New York for providing financial support to carryout this research work.

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