Published in Crop Sci. 44:389-393 (2004).
© 2004 Crop Science Society of America
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CROP BREEDING, GENETICS & CYTOLOGY
Inheritance and QTL Mapping of Antibiosis to Green Leafhopper in Rice
Chunming Wanga,
Hideshi Yasuib,
Atsushi Yoshimurab,
Huqu Zhaia and
Jianmin Wan*,a
a State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China
b Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
* Corresponding author (wanjm{at}njau.edu.cn).
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ABSTRACT
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Nephotettix virescens Distant (green leafhopper: GLH) is one of the major insect pests of rice (Oryza sativa L.) in the temperate rice growing region of Asia. The objective of this study was to determine the chromosome locations of some GLH-resistance genes and conduct genetic analysis of GLH resistance. From a cross between the japonica cultivar Taichung65, which is susceptible to GLH, and indica cultivar ARC10313, which is resistant to GLH, 125 recombinant inbred F10 lines (RIL) were developed. Quantitative trait loci (QTLs) for antibiosis to GLH were detected on chromosomes 3, 5, 11, and 12. The QTL near XNpb292 on chromosome 12 for GLH mortality was from the japonica cultivar, while the other three QTLs on chromosomes 3, 5, and 11 were from the indica cultivar. The percentages of observed phenotypic variance attributable to the major QTLs on chromosomes 3 and 11 were 25.3% and 56.8%, respectively. These two QTLs were close to two green rice leafhopper (GRH, Nephotettix cincticeps Unler) resistance genes, Grh4 and Grh2, respectively. Using NILs (near isogenic lines) of Grh4 and Grh2, we determined that the interaction of these two GRH resistance genes expressed strong resistance to GLH. Four GLH-resistance QTLs, including two major QTLs linked to XNpb144 on chromosome 3 and G1465 on 11, respectively, were identified in this study, and these two major QTLs were located close to Grh2 and Grh4. It may be possible to pyramid these genes to improve resistance to both GRH and GLH.
Abbreviations: GLH, green leafhopper • GRH, green rice leafhopper • MAS, marker-assisted selection • NIL, near isogenic lines • RFLP, restriction fragment length polymorphism • RIL, recombinant inbred line • QTL, quantitative trait locus
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INTRODUCTION
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NEPHOTETTIX VIRESCENS is one of the most serious insect pests of rice. The GLH damages the rice crop directly by sucking the plant sap and indirectly by transmitting viruses such as Rice tungro spherical virus (RTSV) and Rice tungro bacilliform viruses (RTBV), which cause the destructive tungro disease in South and Southeast Asia (Robert and Gunnell, 1992). Pesticides are the common method of controlling an insect pest like GLH, but this method is costly and harms the environment. Resistant cultivars are the most economical and environmentally sound strategy for pest management (Angeles and Khush, 2000). Resistant cultivars are being developed by introgressing resistance genes and pyramiding resistance genes from different origins to increase the durability of resistance (Maliepaard et al., 1995). Molecular markers closely linked to these genes could be used to follow the incorporation of these genes into new cultivars.
Nine GLH-resistance genes have been reported (Kinoshita, 1995; Tomar and Tomar, 1987; Sebastian et al., 1996) but the chromosomal locations of only two genes, Glh3 on chromosome 10 (Sidhu and Khush, 1979) and Glh6 on chromosome 5 (Tomar and Tomar, 1987), are known. Marker-assisted selection (MAS) based on molecular genotypes, such as RFLP (restriction fragment length polymorphism), SSR (simple sequence repeat), and SNP (single nucleic acid polymorphism) holds promise for confirming the selection and accelerating the rate of development of GLH-resistant crops (Harushima et al., 1998; Temnykh et al., 2000; Nasu et al., 2002). The objectives of this study are to (i) conduct a molecular marker-based genetic analysis of GLH resistance by means of an RFLP map and replicated phenotyping experiments, (ii) characterize the GLH resistance of ARC10313, and (iii) determine the modes of the genetic effects of the leafhopper resistance genes.
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MATERIALS AND METHODS
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To investigate the basis of GLH resistance in rice, an RFLP linkage map was constructed with RILs (recombinant inbred lines) as a mapping population. ARC10313 (indica, GLH resistant) was crossed to Taichung65 (japonica, GLH susceptible) as the female parent. One hundred twenty-five F10 lines derived from this cross were developed by SSD (single-seed-descent). The RILs along with the parents were planted in the greenhouse at Kyushu University in 2001. Seven-day-old seedlings of each RIL were evaluated for antibiosis to GLH. Twelve seedlings were transplanted at the Kyushu University farm. Before heading, leaf samples from each RIL were harvested for DNA isolation and RFLP linkage map construction.
The CTAB (cetyltrimethylammonium bromide) method (Rogers and Bendich, 1988) was used with minor modifications for isolating total DNA from rice leaves. DNA was digested with six restriction enzymes (BamHI, BglII, DraI, EcoRI, EcoRV, and HindIII). Southern hybridization was done with horseradish peroxidase-labeled probes using the enhanced chemiluminescence direct nucleic acid labeling and detection system (Amersham Pharmacia Biotech, UK). The RFLP probes were cDNA clones, supplied by the Rice Genome Program, Japan. RFLPs between the parents were surveyed and 113 polymorphic markers were selected to construct the linkage map. The order of the RFLP markers was based on the rice map described by Harushima et al. (1998) and Tsunematsu et al. (1996). Linkage analyses were performed with MAPMAKER/EXP v3.0 (Lander et al., 1987).
NILs were used to enhance the precision of determining the genetic effect of a single QTL. Grh2 and Grh4, which are located on chromosome 11 and 3, respectively, confer resistance to green rice leafhopper (GRH), Nephotettix cincticeps Uhler (Yazawa et al., 1998). Two NILs with a single gene, NIL(Grh2) or NIL(Grh4), and one NIL with both genes, NIL(Grh2Grh4), were developed from a cross between the susceptible cultivar Kinmaze as the recurrent parent and DV85 as the resistance donor parent by MAS (Fig. 1) . The NILs along with the parents were sown in the greenhouse at Kyushu University in 2001 and the seedlings of each NIL were evaluated for antibiosis to GLH.
The GLH population originated from adult insects collected from a paddy field in Fukuoka, Japan and maintained at 25°C with 16 light/8 dark photo regime. To test the antibiosis, 7-d-old seedlings of each RIL and NIL were infested with 7 to 10 second- and third-instar nymphs of GLH for phenotyping GLH resistance. The RIL and NIL seeds were sown in the greenhouse and the seedlings at one-leaf stage were infested with GLH nymphs.
For RIL materials, GLH infestations were conducted with five replications. Each seedling was transferred into a tube, then 7 to 10 nymphs of GLH were transferred into each tube. Three days after infestation, the mortality of the GLH nymphs in each tube was counted, and the mortality of the nymphs in the five tubes of each RIL was averaged and used as the antibiosis rating. The GLH infestation for the NILs had seven replications.
The association between phenotype and marker genotype was investigated by single point analysis (SPA) conducted with QGene and composite interval mapping (CIM) with QTL Cartographer (Nelson, 1997; Zeng, 1994).
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RESULTS
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The RFLP map based on the RILs included 113 RFLP markers on the whole genome scale (Fig. 2)
. On the basis of previous maps (Harushima et al., 1998; Tsunematsu et al., 1996), the markers in this study were well dispersed throughout the whole genome with a total map length of 1462.4 cM and average distance of 12.9 cM between markers. Linkage orientation of the RFLP markers is in agreement with those of the previously constructed maps, indicating it was appropriate to use the cultivars Taichung65 and ARC10313 for mapping QTL.
Seven to 10 insects per seedling distinguished the resistant seedlings from the susceptible ones. Most insects on the resistant plants died within 3 d after infestation, while those on susceptible plants showed normal growth and development. ARC10313 is resistant to GLH with 82.8% nymph mortality, while Taichung65 is susceptible with 10.0% nymph mortality and resistance segregated among their progenies (Fig. 3)
. QTL analysis was conducted by single point analysis (SPA) with a threshold (P < 0.005) to detect the possible QTLs (Soller et al., 1976). By SPA analysis, putative QTLs were detected close to a region defined by two RFLP markers on chromosome 3 and five RFLP markers on chromosome 11. After composite interval mapping, only one QTL was detected on chromosome 3 and one on chromosome 11 (Table 1, Fig. 2). The percentages of observed phenotypic variance attributable to the two QTLs on chromosomes 3 and 11 were 25.3 and 56.8%, respectively. In addition, two QTLs were detected on chromosomes 5 and 12, which explained 8.2 and 9.2% of the phenotypic variance. The resistant allele of the QTL on chromosome 12 originated from the japonica cultivar Taichung65 and contributed to the GLH resistance from japonica cultivars, while the other three QTLs on chromosomes 3, 5, and 11 were from the indica cultivar ARC10313.
It should be noted that both of the QTLs on chromosomes 3 and 11 in this study were major loci for GLH resistance and these two major QTLs were located close to Grh2 and Grh4 (Yazawa et al., 1998; Yasui and Yoshimura, 1999). To date, NIL(Grh2), NIL(Grh4) and NIL(Grh2Grh4) already have been developed by MAS with G1465 and XNpb144, which are linked to Grh2 and Grh4 respectively. These NILs carried introgressions in the target regions of Grh2 and Grh4 because they were developed by MAS with the RFLP markers linked to Grh2 and Grh4. Thus, the possible role of Grh2 and Grh4 in GLH resistance was thus studied by means of these NILs. NILs carrying a single resistance gene, i.e., NIL (Grh2) with the genotype of Grh2/Grh2 grh4/grh4 or NIL(Grh4) with grh2/grh2Grh4/Grh4, are susceptible to GLH. The average nymph mortality on seven replicated experiments was 5.8 and 2.9%, respectively. NIL (Grh2Grh4) carrying both resistance genes, with genotype of Grh2/Grh2 Grh4/Grh4, expressed strong resistance to GLH, with an average nymph mortality for seven replications of 95.1% (Fig. 4)
. These results suggest Grh2 and Grh4 confer strong resistance to GLH through complimentary gene action.
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DISCUSSION
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The main finding of this study is identification of four loci for GLH resistance carried by the resistant parent ARC10313. Of the nine GLH-resistance genes reported previously (Kinoshita, 1995; Tomar and Tomar, 1987; Sebastian et al., 1996), the chromosomal location of only two genes, Glh3 on chromosome 10 (Sidhu and Khush, 1979) and Glh6 on chromosome 5 (Tomar and Tomar, 1987) are known. Since none was found to be located on chromosome 3, 11 or 12, at least three GLH-resistance QTLs in this study should be new GLH-resistance loci. The relationship between the QTL on chromosome 5 in this study and Glh6 (Tomar and Tomar, 1987) needs to be investigated further. QTLs were not detected in other chromosome regions, like Glh3 on chromosome 10 (Sidhu and Khush, 1979). Thus, the GLH resistance of ARC10313 is characterized by the QTLs detected in this study and the other previously identified GLH-resistant genes are not found in this cultivar.
Since NILs were used to confirm target genes and genetic effects with the least background intervention, it is meaningful to develop NILs for correspondent QTLs of GLH resistance (GLH NILs) to assess their real genetic effects and precisely map the resistance genes. According to the study reported previously, Grh2 and Grh4 are GRH resistance genes located on chromosomes 11 and 3 and linked to the RFLP markers G1465 and XNpb144, respectively (Yazawa et al., 1998; Yasui and Yoshimura, 1999). G1465 and XNpb144 are linked to the QTLs of GLH resistance on chromosomes 11 and 3 in this study. Since the two major QTLs detected almost mapped to the same location as the loci of Grh2 and Grh4, respectively, these could be the same genes and probably have broad functions of leafhopper resistance. With the three GRH NILs in this study, it is confirmed that Grh2 and Grh4 confer strong resistance to GLH through complimentary gene action. NILs for the other two QTLs of GLH resistance on chromosome 5 and 12 need to be developed to assess their real genetic effects. The RFLP marker G1465 was recently found to be near to a major BPH (brown planthopper, Nilaparvata lugens Stål) resistance QTL (unpublished data, 2003). The chromosome region around G1465 might be associated with a gene cluster which gives resistance to sucking insects in rice.
Pyramiding of the two genes through molecular breeding can be proposed to improve resistance to not only GRH but also GLH. To combine the two genes, it is necessary to identify plants containing both genes from plants containing either one or the other gene. The most reliable method for producing cultivars possessing more than one resistance gene is to have the genes tagged by molecular markers. Cloning and incorporation of these two genes into susceptible breeding lines by means of molecular-based technology will be useful not only to improve rice cultivars for leafhopper resistance but also to elucidate the mechanism of resistance to GLH and GRH.
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ACKNOWLEDGMENTS
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This study was conducted at Kyushu University, Japan by C. Wang as a visiting scientist from Oct. 2000 to Oct. 2001 and supported by China Scholarship Counsel.
Received for publication July 23, 2002.
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ABSTRACT
INTRODUCTION
