Crop Science Journal of Natural Resources and Life Sciences Education
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Published online 26 August 2005
Published in Crop Sci 45:2044-2048 (2005)
© 2005 Crop Science Society of America
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GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

QTL Mapping of Antibiosis Resistance to Common Cutworm (Spodoptera litura Fabricius) in Soybean

Kunihiko Komatsua,*, Shiori Okudaa, Masakazu Takahashia, Ryoichi Matsunagab and Yoshinori Nakazawaa

a National Agric. Research Center for Kyushu Okinawa Region, 2421 Suya, Nishigoshi, Kikuchi, Kumamoto 861-1192, Japan
b Japan International Research Center for Agricultural Science, 1-1 Ohwashi, Tsukuba, Ibaraki 305-8686, Japan

* Corresponding author (kkomatsu{at}affrc.go.jp)


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The common cutworm (CCW; Spodoptera litura Fabricius; Lepidoptera: Noctuidae) is a major pest of soybean [Glycine max (L.) Merr.] in Japan. Previously, we analyzed a soybean cultivar named Himeshirazu and found that it exhibits a heritable antibiosis resistance to CCW. In this study, we performed quantitative trait locus (QTL) analysis using composite interval mapping (CIM) method to locate the source of the antibiosis resistance of Himeshirazu. We used a previously reported F2 segregating population of susceptible ‘Fukuyutaka’ x resistant Himeshirazu. We constructed a linkage map spanning 2270.4 cM with 146 simple sequence repeat (SSR) markers and a phenotype marker (pubescence color). Two QTLs for antibiosis resistance were detected in linkage group (LG) M. One was located between SSR markers Satt220 and Satt175, with a maximum logarithm of odds (LOD) score of 12.7 and r2 = 0.283. The other was located between Satt567 and Satt463, with a maximum LOD score of 6.8 and r2 = 0.159. The resistant allele of both QTLs was derived from Himeshirazu. One-way ANOVA of the F2 family classified by genotypes of SSR loci linked to each QTL suggested that a significant antibiosis effect was exhibited when both QTLs were homozygous for the resistant allele.

Abbreviations: CCW, common cutworm • CIM, composite interval mapping • cM, centimorgan • LG, linkage group • LOD, logarithm of odds • QTL, quantitative trait locus • RFLP, restriction fragment length polymorphism • SII, standardized insect-growth index • SSR, simple sequence repeat


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE COMMON CUTWORM damages soybean crops in southwestern Japan. Resistant soybean cultivars would reduce both crop losses and the cost of insecticide applications. Breeding of elite soybean cultivars with resistance to CCW would have both environmental and economical benefits.

Programs for breeding pest-resistant soybean cultivars and analysis of the resistance have been pursued in the USA and Japan. Three soybean germplasms (PI171451, Kosamame; PI227687, Miyako White; PI229358, Sodendaizu) were identified as resistant to Mexican bean beetle (Epilachna varivestis Mulsant; Coleoptera: Coccinellidae) more than three decades ago (Van Duyn et al., 1971). The three germplasms are also resistant to lepidopteran pests (Hatchett et al., 1976; Luedders and Dickerson, 1977; Lambert and Kilen, 1984) and have been used as resistance donors in breeding programs (Hartwig et al., 1984, 1990; Bowers, 1990). The genetic basis for the resistance in the three germplasms has been dissected in detail since DNA markers were identifed in the late 1990s. Rector et al. (1998)(1999) located QTLs of antixenosis (nonpreference) resistance to corn earworm (Helicoverpa zea Boddie; Lepidoptera: Noctuidae) on four LGs. They identified three QTLs conferring antibiosis (detrimental effects on insect development) resistance to corn earworm, and estimated a locus on LG-M to be the same as a locus of antixenosis resistance (Rector et al., 2000). The QTL on LG-M controls the major part of the antibiosis resistance of PI229358 (Narvel et al., 2001), and the PI229358 allele has a detrimental effect on insect growth even when combined with the transgene cry1Ac (Walker et al., 2002). Information on QTL locations and effects would contribute to effective programs for breeding insect-resistant soybean cultivars and help explain how the resistance operates. QTL analysis of Arabidopsis thaliana (L.) Heynh. revealed that antixenosis resistance to the herbivorous insect Trichoplusia ni Hubner (Lepidoptera: Noctuidae) depends on the composition of chemicals derived from glucosinolates (Jander et al., 2001; Lambrix et al., 2001). The study of mechanisms of insect resistance in soybean has made little progress so far, but new analytical tactics using molecular markers can now help.

We reported another pest-resistant germplasm named Himeshirazu (Komatsu et al., 2004). Himeshirazu has more effective antibiosis resistance to CCW than ‘Sodendaizu’ (PI229358), and the major part of the resistance is controlled by a locus (or loci) located on LG-M (Komatsu et al., 2004). The position of the locus was not clear, although it appeared to be linked with SSR marker Satt220. The genetic relationship between Himeshirazu and Sodendaizu in the resistance also remained unidentified. To study the resistance of Himeshirazu more closely, QTL analysis was required. Thus, in this study, we performed QTL analysis of the antibiosis resistance of Himeshirazu using an F2 population derived from a cross between Himeshirazu and the susceptible cultivar Fukuyutaka to reveal the QTL position and its effect.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Materials
We used an F2 population developed from a cross between Fukuyutaka and Himeshirazu. A population of 143 plants was evaluated for its antibiosis effect on CCW (Komatsu et al., 2004), and a linkage map was created from the SSR segregation data. Fukuyutaka is a leading cultivar in southwestern Japan and is susceptible to CCW. The parents, F1 seeds, and an F2 population were sown in potting compost on 31 May 2001; 2 weeks later they were transplanted to an experimental field of the National Agriculture Research Center for the Kyushu Okinawa Region. No chemicals were applied for pest control.

Evaluation of the Antibiosis to CCW
The bioassay procedure and the evaluation of the segregating population have been described in detail previously (Komatsu et al., 2004). The bioassay was performed in an air-conditioned room maintained at 25 ± 1°C with a 16-h light/8-h dark photoperiod. Sixth-instar CCW larvae that had been reared on an artificial diet (Insecta LF S; Nippon Nousan Kougyo Co., Yokohama, Japan) until the fifth instar were used for the bioassay. At the end of the fifth instar, each larva was transferred to a plastic cup 6.5 cm wide by 4.0 cm deep. After ecdysis, one or two leaflets were supplied to the cup every day until pupation. We checked larvae for pupation every 8 h. The pupal weight was measured on the fourth day after pupation. The duration of the sixth-instar and pupal weights were used to calculate an index of antibiosis resistance. Because of the different growth patterns between males and females (i.e., male larvae develop into pupae faster than females and are smaller than the females), the durations and weights could not be used directly. To correct for this difference, we developed a standardized insect-growth index (SII). The SII equals the pupal weight divided by the sixth-instar duration. A higher SII indicates that the rearing plant has a lower antibiosis resistance. Six larvae were used per plant, and the average of the six SIIs was used as the index of antibiosis.

Genotyping of the F2 Population
We used 146 SSR markers and a phenotype marker to construct a genetic linkage map. Of the SSRs, 139 were reported by Cregan et al. (1999) or published in the Soybase (http://soybase.org; verified 14 May 2005). The other 7 had been developed by Harada et al. at Chiba University (personal communication, February 2001). The phenotype marker was the pubescence color, governed by the T locus (Woodworth, 1921). DNA was extracted from each plant by the method of Doyle and Doyle (1990). The PCR amplification conditions have been described by Komatsu et al. (2004). For weakly amplified loci, the annealing temperature was lowered to 47.0°C from 53.5°C. The PCR products were electrophoresed in 3.5% agarose gel or 10.0% acrylamide gel (19:1 acrylamide/bis-acrylamide) or 6.0% denaturing acrylamide gel (19:1 acrylamide/bis-acrylamide, 6 M urea). TBE buffer (90 mM of Tris, 90 mM of boric acid, and 2 mM of EDTA) was used for all gels at electrophoresis. Ethidium bromide was used to visualize the banding pattern of the agarose and acrylamide gels. When the band intensity of ethidium bromide stain was weak, we used a Gel-Star kit (TaKaRa, Kyoto, Japan) to obtain a clearer electrophoretogram. For the denaturing acrylamide gel, we used a silver stain method with the Silver Sequence DNA Sequencing System (Promega K.K. Japan, Tokyo, Japan).

Genetic Mapping and QTL Analysis
MAPMAKER/EXP 3.0b (Lander et al., 1987) was used to group and order the loci. The linkage distances were estimated with the Kosambi mapping function (Kosambi, 1944). The minimum LOD score and maximum distance for linkage construction were adjusted to 2.8 and 45.0 cM. We used a CIM method (Zeng, 1993, 1994) to estimate the QTL locations and effects with QTL Cartographer version 1.16 software (Basten et al., 2002). The LOD score criterion for QTL significance was estimated by means of a permutation test (Churchill and Doerge, 1994) with 1000 permutations. The threshold level of the LOD score was set at 3.55 (equivalent to a 5% genome-wise Type I error rate). To confirm the effect of a detected QTL, we performed a general linear model analysis of the F2 population with the genotype of the most closely linked marker to each QTL as the predictor variable and SII as the response variable.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The results of antibiosis evaluation of the F2 population have been reported in detail previously (Komatsu et al., 2004). In brief, the broad-sense heritability of the SII was estimated as 71.3%. The histogram of SIIs of 143 individual plants was continuous, but bimodality was clear: there were two peaks in the histogram, with the higher one on the higher SII (lower antibiosis resistance) side. The frequency ratio of the higher SII (lower antibiosis) part and the lower SII (higher antibiosis) part, divided at SII = 7.4, did not deviate significantly from 3:1 (0.1 < P < 0.2). This result suggests that a recessive gene controls a substantial part of the antibiosis resistance. In addition, a significant difference was found among the SIIs of the three genotypes (homozygous for Fukuyutaka, heterozygous, and homozygous for Himeshirazu) of Satt220, an SSR locus on LG-M. This suggests that the presumed gene is located around the Satt220 locus.

For genetic mapping in the present study, we obtained segregation data on 147 genomic loci in the F2 population. The loci consisted of 146 SSRs and a pubescence color locus, T. These loci formed 23 LGs. The summed linkage distance was 2270.4 cM. The constructed LGs and the arrangement of loci in each LG corresponded well with the previously reported order (Cregan et al., 1999; Harada et al., Chiba University, personal communication, April 2004). However, locus Satt102, previously reported as located on the mid-part of LG-K (Cregan et al., 1999), was located between Satt330 and Satt440 of LG-I. The segregation ratio of Satt102 did not deviate statistically from the expected ratio of 1:2:1. The linkage relationships among the three loci remained if the threshold LOD score for linkage construction was raised to 4.0 in MAPMAKER/EXP. Another genetic mapping program, MAPL98 (Ukai et al., 1995), estimated the position of Satt102 as the same. As there was no reason to exclude Satt102 from map construction, we regarded it as a locus of LG-I.

Using the SIIs and segregation information of the 147 loci, we ran a QTL analysis. CIM revealed two QTLs for antibiosis. They were linked to each other and present on LG-M (Fig. 1). We named the QTL with the higher LOD score CCW-1, and the other CCW-2 (Table 1). CCW-1 was located between Satt220 and Satt175, and CCW-2 between Satt567 and Satt463. CCW-1 had a higher r2 value (the proportion of variance attributed to the QTL in all phenotypic variance). The resistant allele of each originated in Himeshirazu. The resistant allele of CCW-1 is clearly recessive, but almost no dominance effect was observed in CCW-2.



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  		Fig. 1. Logarithm of odds (LOD) scores associated with the standardized insect-growth index (SII) of common cutworm growth on linkage group M, estimated by means of the composite interval mapping method. An LOD score of 3.55 for the QTL detection threshold was associated with a Type I error rate of 5% from a 1000-permutation test.

 

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  		Table 1. QTL for standardized insect-growth index (SII) detected in the F2 population derived from ‘Fukuyutaka’ x ‘Himeshirazu’ by the composite interval mapping method

 
To confirm the effect of a QTL, a general linear model analysis using marker genotype class as the predictor variable and phenotypic data as the response variable is usually performed. In our case, the two QTLs were estimated to be linked to each other. Therefore, to confirm the effect of each QTL separately, we classified the population with two marker loci, each one tightly linked to one QTL. We identified the plants genetically fixed at Satt175 (linked to CCW-1) and Satt567 (linked to CCW-2), and compared SIIs of the four genotypes (Table 2). The object of this analysis was to confirm the effects of each QTL, so the information on the plants heterozygous at both SSR loci was excluded for simplification. Because the genotype class Satt175-Fukuyutaka x Satt567-Himeshirazu had no repetition, we could not use two-way ANOVA. One-way analysis of variance revealed a significant difference between the genotype with the Himeshirazu allele at both loci and the other two genotypes (Table 2). This indicates that when the Himeshirazu alleles of both loci are assembled, significant antibiosis is expressed, although the effects of each QTL and their epistatic interactions are not clear.


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  		Table 2. Standardized insect-growth index (SII) of four fixed genotype classes at two SSR loci linked to the insect resistance QTLs in the F2 population, and of the parents.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Using 146 SSR loci and a phenotype locus, we constructed a linkage map of 23 LGs and spanning 2270.4 cM with the F2 progeny of CCW-susceptible Fukuyutaka and CCW-resistant Himeshirazu. The order of loci agreed closely with the order reported by Cregan et al. (1999), except that locus Satt102 was located on LG-I in our map, instead of on LG-K in their map. We presume that homeologous chromosome regions bring about this difference. Homeologous regions of different soybean chromosomes have been detected via restriction fragment length polymorphisms (RFLPs), qualitative genes, and QTL linkage relationships (Shoemaker et al., 1996; Lohnes et al., 1997). Although no homeologous segment between LG-I and LG-K has been reported, both LGs have an RFLP locus (A510_3 and A510_2) detected by the same probe (Shoemaker and Specht, 1995; Cregan et al., 1999), and Satt102 is clearly linked to A510_2 in the map of Cregan et al. (1999). These facts suggest that the two LGs share a homeologous segment containing the Satt102 locus. Further, Cregan and Quigley (1997) reported the presence of "extra" PCR products amplified with the expected products in SSR analysis. It is possible that some PCR products originating from different but homeologous chromosome segments are amplified by a primer pair. As a result, we believe that the primer pair for Satt102 amplified the locus on the LG-I segment instead of the locus on the LG-K segment in our mapping population. This instance suggests that the map of Cregan et al. (1999) does not always fit completely all mapping populations.

CIM analysis of antibiosis to CCW revealed two QTLs, linked to each other and located on LG-M. The peak LOD score of CCW-1 was detected between Satt220 and Satt175. In insect-resistant PI171458 and PI229358, the major QTL for antibiosis to corn earworm was detected at almost the same position (Rector et al., 2000; Narvel et al., 2001). In addition, the resistant alleles of PI171458 and PI229358 are partially recessive (Rector et al., 2000), as is the Himeshirazu allele (Table 1). These analogies suggest that the antibiosis QTL of the insect-resistant germplasms and CCW-1 of Himeshirazu are identical, although an allelism test would be required to confirm that. The other QTL, CCW-2, was located between Satt567 and Satt463. A weak QTL for the reduction of pupal weight of corn earworm has been detected around Satt567 in the susceptible cultivar Minsoy (Terry et al., 2000). But that QTL did not affect the weight of 12-d-old larvae, and explained only 7% of pupal weight variation. In addition, the population used in that analysis was derived from susceptible cultivars, so there was little phenotypic variation in antibiosis in the segregating population. These facts indicate that the effect of the resistance allele is very limited. No significant QTL for antibiosis has been found around Satt567 and Satt463 in the three insect-resistant germplasms. Whether CCW-2 is identical to the QTL found in Minsoy or not, our analysis has revealed the effective allele of Himeshirazu for the first time.

Little is known about the mechanism of pest resistance in soybean. The involvement of a recessive allele in the antibiosis might be a clue. A locus for antixenosis resistance to a herbivorous insect, Trichoplusia ni, was detected in Arabidopsis, and its resistant allele is nearly recessive (Jander et al., 2001). The gene appears to code an epithiospecifier protein which causes the formation of nitriles and epithionitriles during hydrolysis of glucosinolates (Lambrix et al., 2001). Loss of function of the gene leads to the formation of isothiocyanates, which deter herbivores. It is possible also that some secondary metabolite confers the antibiosis resistance of soybean. For instance, some Arabidopsis mutants with a malfunction in salicylic acid–dependent defense responses to microbial pathogens exhibited a higher level of antibiosis resistance to T. ni than the wild-type (Cui et al., 2002). Although the mechanism has not been revealed in detail, it is clear that the loss of function of the genes causes an increase in antibiosis resistance. The salicylic acid–dependent pathogen or wounding stress response system is considered to be common in higher plants (Reymond and Farmer, 1998), so it is not strange that some disorders of the salicylic acid–dependent system lead to an increase in the antibiosis against herbivorous insects in soybean.

The tandem arrangement of the QTLs for antibiosis is interesting. Clusters of pathogen-resistant gene analogs were detected in soybean (Kanazin et al., 1996). In maize (Zea mays L.), duplicated genes (p1 and p2) control the content of chemicals detrimental to corn earworm (Zhang et al., 2000, 2003). These genes have a highly conserved motif, are tightly linked, and jointly regulate flavone biosynthesis. The soybean genome is considered to have been formed via duplication of an ancestral genome according to both classical evolutional studies and molecular genetic analysis (Shoemaker et al., 1996). Thus, opportunities for genome rearrangements that generate homologous genes might be plentiful. Although the CCW-resistance genes of soybean have not been identified, it would be significant for the study of genome evolution if the QTLs are duplicated homologous genes. Besides the linkage relationship, a notable locus shares the same LG: LG-M contains the gene for vegetative storage protein A (VSP A). The SSR locus marked "VSP A" in Fig. 1 originates from the 5' noncoding region of the soybean VSP A gene, vspA (Harada, personal communication, April 2004). The gene is activated by wounding or jasmonic acid treatment (Mason and Mullet, 1990). In Arabidopsis, a gene homologous to soybean VSP is expressed also by wounding or methyl jasmonate treatment (Berger et al., 1995). Jasmonate plays an important role in resistance to a chewing insect (common fungus gnat, Bradysia impatiens Johannsen; Diptera: Sciaridae) in Arabidopsis, even though the mechanisms are still unidentified (McConn et al., 1997). It is clear that vspA is not the soybean CCW-resistance gene, because the position of the vspA locus differs from the QTL position. But it is also clear that some genes involved in the response to wounding stress are gathered on the same LG of soybean. This phenomenon is interesting not only from the viewpoint of genome evolution, but also for speculation about the mechanism of the antibiosis resistance in soybean.


   ACKNOWLEDGMENTS
 
We wish to thank Dr. Kyuya Harada at Chiba University for providing the primer information of his original SSR loci. We are grateful to Dr. Masao Ishimoto at National Agricultural Research Center for Hokkaido Region for useful comments on the manuscript. We thank Ms. Keiko Uesugi for her excellent technical assistance. This work was supported by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Rice Genome Project DM-1204), for which the authors are very grateful.

Received for publication December 20, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 




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K. Komatsu, M. Takahashi, and Y. Nakazawa
Antibiosis Resistance of QTL Introgressive Soybean Lines to Common Cutworm (Spodoptera litura Fabricius)
Crop Sci., March 19, 2008; 48(2): 527 - 532.
[Abstract] [Full Text] [PDF]

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