Identification of quantitative trait loci associated with small brown planthopper (Laodelphax striatellus Fallén) resistance in rice (Oryza sativa L.)LE QUANG TUYEN1,2, YUQIANG LIU1, LING JIANG1, BAOXIANG WANG1, QI WANG1, THAN THI THU HANH1,2 and JIANMIN WAN1,3

1State Key Laboratory of Crop Genetics and Germplasm Enhancement/Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing, PR China2Institute of Agricultural Sciences for Southern Vietnam, Nguyen Binh Khiem, Ho Chi Minh City, Vietnam3Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, PR China

Tuyen, L. Q., Liu, Y., Jiang, L., Wang, B., Wang, Q., Hanh, T. T. T. and Wan, J. 2011. Identification of quantitative trait loci associated with small brown planthopper (Laodelphax striatellus Fallén) resistance in rice (Oryza sativa L.). – Hereditas 000: 001–008. Lund, Sweden. eISSN 1601-5223. Received 13 June 2011. Accepted 18 October 2011.

F2 and BC1 populations derived from the cross between 02428 / Rathu Heenati were used to investigate small brown planthopper (SBPH) resistance. Using the F2 population, three QTLs for antixenosis against SBPH were located on chromosomes 2, 5 and 6, and accounted for 30.75% of the phenotypic variance; three QTLs for antibiosis against SBPH were detected on chromosomes 8, 9 and 12. qSBPH5-c explaining 7.21% of phenotypic variance for antibiosis was identified on chromosome 5 using the BC1 population. A major QTL, qSBPH12-a1, explained about 40% of the phenotypic variance, and a minor QTL, qSBPH4-a, was detected by the SSST method in both the F2 and BC1 populations. The QTLs indentified in the present study will be useful for marker assisted selection of SBPH resistance in rice.

Jianmin Wan, National Key Laboratory for Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, PR China; Chinese Academy of Agricultural Sciences, Beijing 100081, PR China. E-mail: wanjm@njau.edu.cn

Rice (Oryza sativa L.) is a staple food for more than 50% of the world population. Insect pests are major biotic con-straints in rice production. The small brown planthoper (SBPH), Laodelphax striatellus Fallén (Homoptera: Delphacide), is one of the most destructive and wide spread insect pests throughout the main rice-growing areas in Asia. The adults and nymphs of SBPH are sap-suckers and the infested rice leaves turn yellow, wilt, and eventually die, resulting in yield losses and reduced grain quality. More importantly, the pest also transmits viral diseases, such as rice stripe virus (RSV) (Zhang et al. 2011), and rice black-streaked dwarf virus (RBSDV) (Wang et al. 2010), which often cause more severe yield losses than the actual feeding damage.

Protection against SBPH in past years mostly depended on insecticides. However, chemical control is both costly and environmentally harmful, and can also lead to pesti-cide resistance in the insect, natural enemy death and prob-lems of pesticide residues in rice grain and straw. Resistant cultivars are recognized as the most economic and effec-tive measure for controlling the insect. Resistant varieties are being developed by introgression of resistance genes and pyramiding resistance genes from different origins to increase the durability of resistance. Molecular markers closely linked to resistance genes could be helpful in molecular breeding of new resistance varieties.

So far, only a few SBPH resistance genes have been reported. Three SBPH resistance QTLs, Qsbph2b, Qsbph3d and Qsbph12a on chromosomes 2, 3 and 12 were detected using an F2 population from a cross of Wuyujing 3 (japonica) and Mudgo (indica) (Duan et al. 2009). In another study, backcross inbred lines (BILs) from Nipponbare/Kasalath // Nipponbare, were used to detect QTL for SBPH resis-tance. In the standard seedling-box screening test (SSST), three QTLs (Qsbph3b, Qsbph11d, and Qsbph11e) were mapped on chromosomes 3 and 11. Three QTLs (Qsbph3c, Qsbph8 and Qbph11f ) conferring antixenosis were also detected on chromosomes 3, 8 and 11 (Duan et al. 2010). In addition, two QTLs, Qsbph2 and Qsbph11g on chromosomes 2 and 11, from ‘Kasalath’ expressed antibiosis against the pest (Duan et al. 2008).

One major resistance gene was always short-lived due to adaptation of insect population in overcoming its effect. Quantitative resistance is therefore considered to be more durable. The SriLankan indica rice cultivar Rathu Heenati was shown to be resistant to all four known biotypes of brown planthopper (BPH) (Lakshiminarayana and khush 1977; siDhu and khush 1978, 1979; ikeDa and kaneDa 1981). Three QTLs Qbph3, Qbph4 and Qbph10 for resistance to this pest were detected on chromosomes 3, 4 and 10, and a major QTL, Qbph4, was designed as Bph17 (sun et al. 2005). The same variety was found to

Hereditas 000: 001–008 (2011)

© 2011 The Authors. This is an Open Access article. DOI: 10.1111/j.1601-5223.2011.02231.x

2 L. Q. Tuyen et al. Hereditas 000 (2011)

be also resistant to SBPH (Duan et al. 2008). In order to find more SBPH resistance genes, the object of this study was to identify resistance genes in rice variety Rathu Heenati.

MATERIAL AND METHODS

Plant material

Indica variety Mudgo and Japonica variety Wuyujing 3 were used as resistant and susceptible controls, respec-tively. Mapping populations of 162 F2 and 150 BC1 plants were derived from a cross of 02428 and Rathu Heenati (RH) and the backcross 02428 / Rathu Heenati // 02428; each F2 and BC1 individual was self-fertilized to obtain sets F2:3 and BC1F2 lines.

Small brown planthoppers

The small brown planthopper population used in the study was collected from rice fields in Nanjing, and maintained on wheat for four generations before being transferred to rice Wuyujing 3 under greenhouse conditions at Nanjing Agricultural University. The insects were confirmed to be non-viruliferous by a dot-immunobinding assay and PCR detection (Qin et al. 1994).

Evaluation of SBPH resistance

Modified standard seedling-box screening test (SSST)Soil was transferred to plastic cups 8 cm in diameter and 9 cm in height with and a hole in the base. All seeds were germinated at 30°C. Thirty germinated seeds per F2:3 or BC1F2 line were planted in the soil. Twenty eight cups, including resistant and susceptible control varieties, were randomly placed in a 65 44 14 cm plastic seedling-box and kept in water 2 cm deep. There were three replicates for each cultivar and line. Seedlings were grown in a greenhouse under natural light and ventilation conditions at 25 1°C.

Fifteen small brown planthopper nymphs per plant (2nd and 3rd instars) were placed on each seedling at the 1.5–2 leaf stage. Scoring was done when approximately 90% of Wuyujing 3 seedlings had died, each line or vari-ety was assigned a numerical score between 0 and 9 following a standard evaluation system used at IRRI (1988). The mean value of three replicates was as the damage score.

Antixenosis testFifteen germinated seeds per line were sown to soil in plastic seedling-boxes; two rows per line. Seedlings were grown under natural light at 25 1°C in the green-house. At the 1.5–2 leaf stage, the seedling-boxes were transferred into cages covered with nylon nets, and then

seven (2nd–3rd instar) small brown planthopper nymphs were placed on each seedling. There were three replicates for each cultivar and line.

The number of insects on each seedling was counted 24 h after infestation, and then dispersed evenly among the seedlings after counting every day. The average num-ber of insects on each line was calculated and treated as the antixenosis value after 5 days scoring.

Antibiosis testColorless 7 cm diameter and 17 cm high glass cups were used in this experiment. A hot solution (30 ml), contain-ing 1 g agar, 100 ml H2O and 15 ml MS, was poured into each cup, and 5 germinated seeds were planted after the culture medium had cooled. There were three replicates for each cultivar and line. Seedlings were grown in the greenhouse at 25 1°C under natural light.

At the 1.5–2 leaf stage, 30 small brown planthopper nymphs (2nd–3rd instar) were placed on each seedling in each cup which was then covered by nylon net. The survival of nymphs in each cup was recorded each day for 10 days, and the average survival of insects on each line was calculated and treated as the antibiosis value.

Statistical analyses

Statistical analysis of data were performed using one- way ANOVA and significant differences were identified by the LSD test at P 0.05 using Excel ver. 2003 and SPSS Statistics ver. 17.0. c2-tests were performed to examine the goodness of fit between expected Mendelian ratios and SSR and phenotype data.

DNA extraction

Two to three hundred mg fresh leaves of each F2 and BC1 plant (and parents) were collected and put in a pre-cooled mortar. Liquid nitrogen was added and the tissue was ground to fine powder with a pestle and transferred into a 2.0 ml Eppendorf tube; 600 ml of extraction buffer (100 mM Tris-HCl pH 8.0, 50 ml EDTA-Na2, 500 mM NaCl, 1.25% SDS) were added and to the Eppendorf tube and the mix-ture was homogenized. The tube was incubated in a 65°C water bath for 30 min and shaken 3–4 times until the sam-ple turned deep green. One hundred ml of KAC (5 mol l21) solution was added, homogenized and incubated on ice for 30 min; 300–400 ml of chloroform:isoamyl alcohol (24:1) solution was added and shaken at 120 rpm for 30 min; centrifuging was done at 12 000 rpm for 5 min, and 200 ml of aqueous solution at the top was transferred to a new Eppendorf tube; 400 ml of chilled 99.9% ethanol (220°C) was added and incubated at 220°C for 20 min. Centrifuging was done at 12 000 rpm for 5 min; the super-natant decanted and washed with 70% alcohol to dry the

Hereditas 000 (2011) Trait loci associated with planthopper resistance in rice 3

Table 1. Responses of parents and control varieties to small brown planthopper. Sixty seedlings were used in each test. HR - highly resistant, HS - highly susceptible.

Variety Response (mean SE) Description

Rathu Heenati 1.2 0.12 HRMudgo 1.5 0.20 HR02428 8.5 0.20 HSWuyujing 3 8.3 0.24 HS

Fig. 1a–c. Frequency distribution of SBPH resistance in the 02428 / RH F2 population. (a) seedling-box screening test; (b) antixenosis test; (c) antibiosis test.

DNA. The DNA was re-suspended and dissolved in 100 ml of TE, and stored at 4°C.

Amplification of microsatellites and detection of polymorphism

SSR markers and genomic sequences were obtained from the Gramene database (www.gramene.org/marker/index.html) (mccouch et al. 2002) SSR analysis was performed following the procedure of Chen et al. (1997) with minor modifications. Amplifications were performed

in a 10 ml system containing 10 mmol l21 Tris-HCl pH 8.3, 50 mmol l21 KCl, 1.5 mmol l21 MgCl2, 50 mmol l21 of each dNTP, 0.2 mmol l21 each of primers, 0.5 U Taq poly-merase (TaKaRa, Dalian) and 20 ng of DNA template, using a PTC-200 thermal cycler programmed at 95°C for 5 min, followed by 34 cycles of 30 s at 94°C, 30 s at 55°C, 40 s at 72°C and a final extension of 10 min at 72°C. Amplified DNA products were electrophoresed on 8% polyacylamide non-denaturing gels in 0.5 TBE buffer and detections were made by the silver staining method (sanguinetti et al. 1994). The bands were scored on a light box lit with fluorescent lamps.

SSR mapping and QTL detection

Linkage groups were assembled using MAPMARKER 3.0 software at an LOD score of 3.0 (LincoLn et al. 1992). QTLs were detected by composite interval mapping (CIM) using Windows QTL Cartographer 2.5 software with LOD threshold of 2.2 and enabled significance of 0.05 (Basten et al. 2005, www.statgen.ncsu.edu/ qtlcart ).

4 L. Q. Tuyen et al. Hereditas 000 (2011)

the 161 SSR markers; the average distance between markers was 11.8cM. A second map constructed for data from the 150 BC1 lines covered 1, 720.7cM with an average marker interval of 10.7 cM. The orders of mark-ers in the two maps were similar and in agreement with previously published maps (temnykh et al. 2000; mccouch et al. 2002).

Identification and mapping SBPH resistance gene

The standard seedling-box screening test (SSST) and QTL identificationThe parents and control varieties were tested for SBPH response by the SSST method. The resistance values of Rathu Heenati and 02428 were 1.2 0.12 and 8.5 0.20, respectively; confirming that Rathu Heenati was highly resistant to SBPH and 02428 was highly susceptible. Rathu Heenati was more resistant than the resistant con-trol, Mudgo (Table 1, Fig. 1a).

The F2 plants were classified into three classes: homozy-gous resistant (RR) for Rathu Heenati alleles, homozy-gous susceptible (rr) for 02428 alleles, and heterozygous (Rr). In the F2 population, SBPH resistance scales were

RESULTS

Construction of linkage map

A total of 744 SSR markers, distributed across all 12 chro-mosomes, were used in a parental survey of polymorphism between Rathu Heenati and 02428. One hundred and sixty one markers (21.6% of all markers) polymorphic between the parents were used to assay the 162 F2 lines and 150 BC1 lines and to construct the molecular linkage map.

Using 162 F2 lines from 02428 / Rathu Heenati, a mole-cular linkage map of 1907.7cM was constructed with

Table 2. Segregation of small brown planthopper resistance in F2 population derived from 02428 / Rathu Heenati. c2

1:2:1 value for 1RR: 2Rr: 1rr 4.85 c20.05, 2

5.99.

F2 genotype

No. of F2 individuals

Phenotype of corresponding F2:3 families

RR 48 RS 3Rr 67 3 RS 6rr 47 RS 6

Fig. 2a–c. Frequency distributions of the SBPH-resistance in BC1 population. (a) seedling-box screening test; (b) antixenosis test; (c) antibiosis test.

Hereditas 000 (2011) Trait loci associated with planthopper resistance in rice 5

The damage scores of 150 BC1F2 lines infested with the insect ranged from 0.3 to 9.0 showing an apparent valley being around 6 in the distribution curve (Fig. 2a) and the separation rate of resistance and susceptible plants fits 1:1 (66:84) ratio (c2 2.16 c2

0.05; 1 3.84) (Table 3). These results indicated that a major dominant gene controlled the SBPH resistance in Rathu Heenati.

Five QTLs for SBPH resistance, designated qSBPH1-a, qSBPH2-a, qSBPH4-a, qSBPH12-a1 and qSBPH12-a2 were detected by 162 F2 lines (Fig. 3). In which, qSBPH1-a with LOD score of 4.45 was detected at location between RM323 and RM522 on the short-arm of chromosome 1, explaining 7.62% of phenotypic variance (PVE). qSBPH2-a at region of RM525-RM5916 on the long-arm of chromosome 2, with LOD scores of 2.40 and 4.20% of PVE; qSBPH4-a at location between RM451 and RM5473 on the long-arm of chromosome 4, with PVE is 9.08% and LOD score 3.77; qSBPH12-a2 on the short-arm of chro-mosome 12 at location between RM6288 and RM6296,

distributed to a continuous range of 0.6 to 9.0, with the mean value 5.1, and two valleys appeared in 4 and 7 (Fig. 1a). According to the score criterion in the seedling bulk test by IRRI (IRRI 1996), the plants with scales within the ranges of 0–3, 3.1–6.0 and 6.1–9.0 were classi-fied into three types as high resistance, moderate resis-tance and susceptible (Table 2). The segregation of the F2 population showed a good fit of 1RR:2Rr:1rr (c2 4.85 c2

0.05; 2 5.99) (Table 2).

Table 3. Segregation of small brown planthopper resistance in BC1 population derived from 02428 / Rathu Heenati // 02428. c2 value for 1Rr:1rr is 2.16 (c2

0.05:1 3.84).

BC1 genotype

No. of BC1 individuals

Phenotype of corresponding BC1F2 families

Rr 66 RS 6rr 84 RS 6

Fig. 3. QTLs for SBPH resistance detected in F2 and BC1 populations by three phenotypic screening methods.

6 L. Q. Tuyen et al. Hereditas 000 (2011)

Three QTLs of antixenosis against SBPH were detected by 162 F2:3 lines, such as qSBPH2-b1 at location between RM213-RM535 on the long-arm of chromosome 2, with a LOD score of 3.46 and 11.46% of PVE of antixenosis against SBPH; qSBPH5-b at region between RM592-RM574 on the chromosome 5, with a LOD score of 2.28 and PVE is 9.52%, and qSBPH6-b at location between RM30 and RM439 on the long-arm of chromosome 6, with a LOD score of 3.74 and PVE of 9.77% (Table 4, Fig. 3).

Another QTL conferring antixenosis against SBPH, qSBPH2-b2 was mapped at the region between RM318-RM240 on chromosome 2 by 150 BC1F2 lines, with LOD score of 2.28 and 6.6% of the phenotypic variance in this population, but indicated by the additive effect, resistant alleles at qSBPH2-b2 did not come from Rathu Heenati, but came from 02428 (Table 5, Fig. 3).

Antibiosis test and QTL mapping

The antibiosis values of Rathu Heenati and 02428 were 52.5% and 84.75%, respectively. The survival rate of nymphs were ranged from 52 to 92.67% in the F2 popula-tion, and ranged from 41.27 to 89% in the BC1 popula-tion. The results showed that minor genes controlling antibiosis resistance (Fig. 1c, 2c). A total of three QTLs, designated qSBPH8-c, qSBPH9-c and qSBPH12-c, con-ferring antibiosis against SBPH were detected on chromo-somes 8, 9 and 12 using 162 F2:3 lines, in the region of RS413-RM4085, RM105-RM566 and RM277-RM519

with LOD score of 2.57 and 4.47% of PVE; and the major QTL namely qSBPH12-a1, was detected in loca-tion between RM519 and RM3331 on the long-arm of chromosome 12, with a high LOD score of 18.99 and it explained 37.84% of the phenotypic variance in F2 population. As indicated by the additive effect, resistant alleles at qSBPH1-a, qSBPH2-a, qSBPH4-a, qSBPH12-a2 and qSBPH12-a1 came from ‘Rathu Heenati’ (Table 4).

Two QTLs, qSBPH4-a and qSBPH12-a1 were also identified at the same region of RM451-RM5473, and RM519-RM3331 using BC1 population (Fig. 3), with LOD scores of 2.72, 34.96 and QTLs explained 1.49 and 40.04% of the phenotypic variance respectively. The two QTLs also came from Rathu Heenati (Table 5). These results indicated that a major QTL qSBPH12-a1 and some minor QTLs controlled Small brown planthopper resis-tance in Rathu Heenati.

Antixenosis test and QTL detection

The antixenosis values of Rathu Heenati and 02428 were 3.5 and 6.8, respectively. In the F2 population, the values of antixenosis showed a continuous range of 2.9 to 10.2 in the distribution curve (Fig. 1b). A continuous seg-regation was also observed in BC1 population with a range of insect number from 3.4 to 10.7 and a valley of 6.0 in the distribution curve (Fig. 2b). Both of the normal distri-bution of antixenosis values indicated that minor genes governed antixenosis resistance.

Table 4. QTLs for SBPH response in the 02428 / Rathu Heenati F2 population determined by three phenotypic screening methods.

Phenotyping methods QTL Chromosome Marker interval LOD score PVE (%) Additive effect

Modified standard seedling-box screening test (SSST)

qSBPH1-a 1S RM323–RM522 4.45 7.62 20.81qSBPH2-a 2L RM525–RM5916 2.40 4.2 20.70qSBPH4-a 4L RM451–RM5473 3.77 9.08 21.07qSBPH12-a2 12S RM6288–RM6296 2.57 4.47 20.67qSBPH12-a1 12L RM519–RM3331 18.99 37.84 22.13

Antixenosis test qSBPH2-b1 2L RM213–RM535 3.46 11.46 20.67qSBPH5-b 5S RM592–RM574 2.28 9.52 20.61qSBPH6-b 6L RM30–RM439 3.74 9.77 20.84

Antibiosis test qSBPH8-c 8S RS413–RM4085 2.49 8.42 11.80qSBPH9-c 9S RM105–RM566 2.39 5.70 10.10qSBPH12-c 12L RM277–RM519 2.56 7.75 212.52

Table 5. QTLs for SBPH resistance detected in 02428 / Rathu Heenati // 02428 the BC1 population by three phenotypic screening methods. PVE - % of variance explained.

Phenotyping method QTL Chromosome Marker interval LOD score PVE (%) Additive effect

Seedling-box screening test qSBPH4-a 4L RM451–RM5473 2.72 1.49 20.71qSBPH12-a1 12L RM519–RM3331 34.96 40.04 23.91

Antixenosis test qSBPH2-b2 2L RM318–RM240 2.28 6.61 0.80Antibiosis test qSBPH5-c 5L RM3838–RM3663 2.34 7.21 26.12

Hereditas 000 (2011) Trait loci associated with planthopper resistance in rice 7

RM463 and STS marker S15552 in introgression line IR65482-7-216-1-2 derived from O. australiensis (Jena et al. 2006). Bph21 was also detected between markers RM3726 and RM5479 on chromosome 12L in IR71033-121-15 (rahman et al. 2009) and Duan et al. (2009) dis-covered major QTL Qsbph12a linked tightly to markers I12-17 and RM3331 in an F2 population from Mudgo / Wuyujing 3. On the basic SSR map in the Gramene data-base all these SBPH and BPH resistance genes (bph2, Bph9, Bph18 (t), Bph21 (t) and Qsbph12a) appear to located in the same region between RM519 and RM3331 on chromosome 12L. These results collectively indicate that the region RM519–RM3331 may be involved in response to piercing-sucking insects and could be an inter-esting source of resistance to SBPH, BPH and RBSDV amenable to marker-assisted selection.

Acknowledgements – Le Quang Tuyen and Yuqiang Liu contrib-uted equally to this work. This research was financed by NSFC (31000697), the PhD Programs Foundation of Ministry of Edu-cation of China (20100097120036), Natural Science Foundation of Jiangsu Province of China (BK2010442), National Key Transform Program (2009ZX08009-046B, 2008ZX08001-001, 2008ZX08001-002), the Project of high Biotechnology in Agriculture of Vietnam, Ministry of Agriculture and Rural development, Jiangsu Science and Technology Development Program (BE2009301-3) and Agriculture Science and Technol-ogy Innovation Program in Jiangsu Province (CX(10)131).

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with LOD scores of 2.49, 2.39 and 2.56, respectively. These QTLs explained 21.28% of the total phenotypic variance in the F2 population (Table 4, Fig. 3). While, using 150 BC1, only one QTL, qSBPH5-c with LOD scores of 2.34 and 7.21% of the phenotypic variance was mapped at region between RM3838-RM3663 on the chromosome 5 (Table 5, Fig. 3).

DISCUSSION

Feeding by small brown planthoppers can cause direct damage to rice plants, but more harmful effects come from the two viral diseases, rice stripe virus (RSV) and rice black-streaked dwarf virus (RBSDV) that they trans-mit. Several RSV resistance genes have been indentified, such as Stvb-i, qSTV TQ and qSTV KAS (hayano et al. 2000; Wu et al. 2010; Zhang et al. 2011), and use of these resis-tance genes in rice breeding has reduced losses caused by RSV. RBSDV was only a sporadic disease in some rice-growing areas before 2004, but has become a more serious problem now. To date no source of RBSDV resis-tance has been found. Thus SBPH resistance will not only help to control SBPH, but may be a workable pathway for reducing losses caused by RBSDV.

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