Development of a Sequence Characteristic Amplified ...

Mol. Cells, Vol. 25, No. 2, pp. 205-210

Molecules and Cells ©KSMCB 2008

Development of a Sequence Characteristic Amplified Region Marker linked to the L4 Locus Conferring Broad Spectrum Resistance to Tobamoviruses in Pepper Plants Hyun Jung Kim1, Jung-Heon Han2,4, Jae Hyoung Yoo3, Hwa Jin Cho3,†, and Byung-Dong Kim1,2,4,* 1

Department of Plant Sciences, Seoul National University, Seoul 151-921, Korea; Center for Plant Molecular Genetics and Breeding Research, Seoul National University, Seoul 151-921, Korea; 3 Joongbu Breeding and Research Station, SeminisKorea, Eumsung 369-832, Korea; 4 Research Institute for Agriculture and Life Science, Seoul National University, Seoul 151-921, Korea. 2

(Received July 5, 2007; Accepted December 3, 2007)

To develop molecular markers linked to the L4 locus conferring resistance to tobamovirus pathotypes in pepper plants, we performed AFLP with 512 primer combinations for susceptible (S pool) and resistant (R pool) DNA bulks against pathotype 1.2 of pepper mild mottle virus. Each bulk was made by pooling the DNA of five homozygous individuals from a T10 population, which was a near-isogenic BC4F2 generation for the L4 locus. A total of 19 primer pairs produced scorable bands in the R pool. Further screening with these primer pairs was done on DNA bulks from T102, a BC10F2 derived from T10 by back crossing. Three AFLP markers were finally selected and designated L4-a, L4-b and L4-c. L4-a and L4-c each underwent one recombination event, whereas no recombination for L4-b was seen in 20 individuals of each DNA bulk. Linkage analysis of these markers in 112 F2 T102 individuals showed that they were each within 2.5 cM of the L4 locus. L4-b was successfully converted into a simple 340-bp SCAR marker, designated L4SC340, which mapped 1.8 cM from the L4 locus in T102 and 0.9 cM in another BC10F2 population, T101. We believe that this newly characterized marker will improve selection of tobamovirus resistance in pepper plants by reducing breeding cost and time.

†

Present address: Biotechnology Center, Nong Woo Bio Co., Ltd., 537-17, Yeoju 469-885, Korea.

* To whom correspondence should be addressed. Tel: 82-2-880-4933; Fax: 82-2-873-5410 E-mail: [email protected]

Keywords: Amplified Fragment Length Polymorphism (AFLP); Bulked Segregant Analysis (BSA); Capsicum; L4; Marker Conversion; Sequence Characterized Amplified Region (SCAR); Tobamoviruses.

Introduction Tobamoviruses such as pepper mild mottle virus (PMMoV), tobacco mosaic virus (TMV), and tomato mosaic virus cause severe damage to plants of the Solanaceae family by affecting growth rate and fruit quality. In pepper plants, PMMoV is considered one of the most problematic viruses due to the ease with which it is transmitted in plants by irrigation (Choi et al., 2004), and in contaminated soil (Pares and Gunn, 1989) and seed (Han et al., 2001a; 2001b; Ikegashira et al., 2004). Two strains of PMMoV have been reported in several countries cultivating various pepper plants (Toyoda et al., 2004). PMMoV can be divided into two pathotypes, P1.2 and P1.2.3, according to the symptomatic reactions they elicit on hosts possessing one or other of the homozygous alleles (L+, L1, L2, L3, and L4) at the L locus (Boukema, 1980). The L locus is located near the telomere of the pepper linkage group corresponding to the quantitative trait loci for resistance to Phytopthora capsici, and cucumber mosaic virus resistance on tomato chromosome 11 (Ben et al., 2001). The L gene is accompanied by induction of the hypersensitive response (HR), characterized by rapid cell death, which prevents the virus from spreading within and around the necrotic lesions. L3-mediated resistance does not allow the invasion of pathotype P1.2, which has been the most common pathotype in pepper fields and greenhouses. Because pathotype P1.2 can be effectively controlled by introgressing the L3

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SCAR Marker of Chili Pepper L4 Locus

Fig. 1. Hypersensitive response (HR) of Capsicum chacoense possessing the L4 allele against the pathotype 1.2.3 of pepper mild mottle virus.

allele into commercial pepper cultivars, pathotype P1.2.3 is considered a more serious pathogen than pathotype P1.2. Previously, the L4 allele identified in C. chacoense PI260429 was reported to confer resistance to pathotype P1.2.3 and to provide a broad spectrum of resistance to all tobamovirus pathotypes in pepper plants (Boukema, 1980). The response mediated by the L4 allele was induced by an elicitor, the tobamovirus coat protein, leading to induction of cell death, and eventually limiting the viruses to the infected cells (Gilardi et al., 2004) and evoking the hypersensitive response (HR) (Fig. 1). New pepper cultivars possessing the L4 allele have become available recently in seed markets; they were developed by conventional breeding methods. More introgression of the L4 allele will be required in future to protect peppers from invasion by tobamoviruses. However, adaptation of the L4 allele to new pepper cultivars is a time consuming step if one only uses a conventional breeding strategy. More recently, the application of marker-assisted selection (MAS) to molecular breeding has been used as an alternative or additional tool for breeding plants for resistance to various pathogens including cucumber mosaic virus (Bene et al., 2001), tomato spotted wilt virus (Moury et al., 2000) and potato virus Y (Caranta et al., 1999). In this study we used bulked segregant analysis (BSA) combined with amplified fragment length polymorphism (AFLP) methodology to develop markers linked to the L4 allele. One of the AFLP markers linked to L4 was also converted into a sequence characterized amplified region (SCAR) marker and designated L4SC340. We expect that this marker will be an important tool for improving breeding tobamovirus resistance in commercial seed production by reducing the cost and time required for screening for virus resistance.

Materials and Methods Plant materials Capsicum chacoense ‘PI260429’ carrying the

L4 allele was the pollen donor plant. Maternal plants were Capsicum annuum ‘GCH4’ and ‘SCR’, two elite lines used for commercial F1 seeds and susceptible to TMV pathotype 1.2.3. Two BC4F2 populations were generated from the crosses of GCH4 (for T4) or SCR (for T10) with PI260429. A progeny of T4 with the heterozygous L4 allele was successively backcrossed into GCH4, resulting in a T101 (BC10F2) population of 106 individuals, which was developed by self-pollination of a BC10F1 containing a heterozygous L allele. The other BC10F2 population, designated T102, consisted of 112 individuals and was spontaneously developed through backcrossing a progeny of T10 with a heterozygous L4 allele to HPC using the same strategy as for the T101 population. F3 seeds were harvested from each F2 individual of the T101 and T102 populations by self-pollination and used for determining genotypes for the L4 allele in the F2 individuals. For scoring phenotypes of the L4 allele and harvesting leaf samples, these F2 and F3 plants were grown in a greenhouse at the SeminisKorea Breeding Research Institute, Eumseong, South Korea. Virus inoculation and DNA pooling An inoculum was prepared from leaves of GCH4 infected with pathotype 1.2.3 of pepper mild mottle virus as described by Han et al. (2001a). Two cotyledons of seedlings per individual from T101, T102, and their F3 populations were dusted with carborundum powder and inoculated by gently rubbing with a cotton stick wetted with inoculum. The presence or absence of a hypersensitive reaction on the cotyledons was carefully observed by eye over 2 weeks. Genomic DNA was isolated from each individual using a NucleoSpin DNA Purification Kit (Macherey, Germany). Each DNA sample was adjusted to 50 ng/µl using a DyNAQuant 200 fluorometer (Hoefer, USA). Two DNA bulks, L4/L4 and L+/L+, from each F2 population were prepared by mixing equal volumes of gDNA. Amplified fragment length polymorphism (AFLP) analysis AFLP analysis was performed as described by Vos et al. (1995) with 512 primer combinations consisting of 256 EcoRIA + 3/MseIC + 3 and 256 EcoRIA + 3/MseIG + 3 primer pair combinations. For visualization, EcoRI primers were end-labeled with [γ-32P] ATP. The second PCR product was separated on a 6% denaturing polyacrylamide gel in 1× TBE buffer. The gel was dried with a Gel Dryer (Amersham Pharmacia Biotech, USA) and exposed to X-ray film (Fuji Film, Japan) at −80°C for 1−2 d. The film was developed with a Hyperprocessor (Amersham Life Science, USA). Conversion of AFLP markers into SCAR markers AFLP bands of interest were excised from the dried 6% sequencing gel with a sharp and clean razor blade. The fragment was transferred into a 1.5 ml Eppendorf tube containing 500 µl of distilled water, kept at 4°C for at least 4 h, and centrifuged at 12,000 rpm for 1 min. The supernatant containing the DNA was transferred into a new Eppendorf tube, mixed with the same volume of isopropanol, incubated at −20°C for 3 h, and centri-

Hyun Jung Kim et al.

A

B

Fig. 2. Autoradiograph of BSA-AFLP analysis with primers generating polymorphisms between resistant (R) and susceptible (S) DNA bulks from the T102 population. A. The 19 primers identified from prior screening of T10 DNA bulks were further tested for polymorphism in new DNA bulks of the T102 population. B. Autoradiographs of AFLP screening for recombination among two L4-M markers and the L4 locus. Resistant (R) and susceptible (S) individuals from the T10 and T102 populations were tested.

fuged for 18 min at 12,000 rpm. The pellet was washed twice with 70% ethanol and air dried. It was then dissolved completely in 50 µl of distilled H2O and used for sequencing. Following AFLP analysis with the same primers, the amplified DNA was separated by electrophoresis on a 0.8% agarose gel. The fragment cut from the gel was isolated with a Zymoclean Gel DNA Recovery Kit (Kyongshin Scientific Co. Ltd., Korea) and cloned into the Easy pGem-T Vector (Promega, USA), and the insert DNA was sequenced using an ABI3700 DNA sequencer (Applied Biosystems, USA) at the National Instrumentation Center for Environmental Management at Seoul National University. Primer sets were designed using the Primer 3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) for development of SCAR markers (Genetics Computer Group Inc., USA). The PCR mixture was composed of 3 μl of DNA, 2 μl of 10× PCR buffer (TaKaRa), 0.3 μl of 10 mM dNTP mixture (TaKaRa), 0.2 μl of 5 U/μl Taq polymerase (Roche, USA), 1 μl of 10 pmol/μl each primer, and 14.5 μl of distilled water. PCR was carried out on a PCR machine (PTC-200, MJ research, USA) with the following parameters: denaturation at 94°C for 3 min, and 29 cycles of 94°C for 30 s, 65°C for 30 s, 72°C for 1 min, and 72°C for 10 min as a final extension. PCR products were separated on a 1% agarose gel containing EtBr and visualized under UV light. Linkage analysis Marker linkage analysis was performed using Mapmaker program 3.0b (Lander et al., 1987) at a logarithm of odds (LOD) value of 4.0 and 30 cM of maximum distance, using the mapping function of Kosambi (1944).

A

207

B

Fig. 3. Segregation analysis and mapping of AFLP markers on the pepper T102 F2 population. A. Fragment patterns of the AFLP markers L4-a, L4-b, and L4-c. B. Linkage map generated with MAPMAKER showing the linear order of AFLP markers. All the linkage distances were calculated according to Kosambi (Kosambi 1994). L4 is the morphological marker.

Results BSA-AFLP Two DNA bulks consisting of five resistant (L4/L4) or six susceptible (L1/L1) individuals from the T10 population were screened for polymorphism with 512 primer combinations. Nineteen primer pairs giving scorable bands were selected for further study (data not shown). These primer pairs were used in a screen for polymorphism between resistant and susceptible DNA bulks, each of which consisted of ten homozygous individuals of T102. Three polymorphic bands were found in combinations between EcoRI + aac and either MseI + aat, MseI + aag, or MseI + att; these bands were designated L4-a, L4b and L4-c, respectively (Fig. 2A). In further screening of individual DNAs of each DNA bulk from T10 and T102, primer combinations for L4-a and L4-c allowed detection of one recombinant event for their susceptible individuals. However, no recombinant event was detected in most individuals screened with the primer combination for L4b (Fig. 3B). Linkage analysis A morphological marker for the L4 locus was first generated by calculating genotypes on phenotype data for TMV resistance, in which there was a minor difference of 0.1 cM between the values derived from the F2 and F3 phenotype data (data not shown). For linkage with the L4 locus, a total of 112 F2 individuals from T102 were screened with these primer pairs (Fig. 3A). The distances of these AFLP markers from the L4 morphological marker ranged from 2.5 to 4.5 cM (Fig. 3B). SCAR marker development To transform the L4-b marker into a PCR-based marker for easy scoring on agarose gels, the 314-bp PCR fragment from an acrylamide gel was cloned into the TA-cloning vector (Invitrogen, USA). A homology search in the NCBI data base (http://www.ncbi.nlm.nih.gov/BLAST/) showed no significant match using BlastN, whereas 85% identity to CTV.20 at 4E-39 was observed using tBlastX (Table 1). After designing primer pairs from the sequence of L4-b,

SCAR Marker of Chili Pepper L4 Locus

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Table 1. Description of L4-linked AFLP markers L4-a, b, and c and the derived SCAR marker. Marker designation

Primer combination

Product size (bp)

Blast X (E-value)

Distance from L4 locus (cM)*

L4-a

EcoRI-ATA + MseI-CGC

840

SocE [Myxococcus xanthus] (2e-19)

4.5

L4-b

EcoRI-AGA + MseI-CCA

310

CTV.20 (4e-39)

2.5

L4-c

EcoRI-ATA + MseI-CAC

150

-

5.1

L4SC340

F 5′-AAGGGGCGTTCTTGAGCCAA-3′ R 5′-TCCATGGAGTTGTTCTGCAT-3′

340

CTV.20 (4e-39)

1.8

* Distance was evaluated on 112 F2 individuals of the T102 population.

PCR was performed in the resistant and the susceptible DNA bulks from T101 and T102 individuals. Additionally, three known markers for TMV resistance were tested in the same DNA pools. A single fragment corresponding to the expected 340-bp size was amplified only in the resistant bulks and was designated L4SC340 (Fig. 4A). PMFR11 and PMFR21 (Matsunaga et al., 2003), and WA31-1500S (Sugita et al., 2004) did not show any polymorphism in the R and S bulks of the T101 and T102 populations (Figs. 4B, 4C, and 4D). The L4SC340 marker was further tested in the T101 and T102 populations to elucidate the relationship between genotype and phenotype for tobamovirus resistance. This marker cosegregated with the L4 locus except for two recombinant events that were found in 112 individual DNA samples from T102, whereas only one recombinant was observed in 104 individual DNA samples from T101 (Fig. 4E). The expected distance of the SCAR marker from the L4 locus was 0.9 cM in T101 and 1.8 cM in T102 with a standard error of 0.1 cM for both. Pattern of amplification of the SCAR marker in pepper differentials for the Tobamovirus pathotype To further study L4SC340, the SCAR marker was amplified from the gDNA of five plants (ECW, Tisana, Tabasco, PI159236, and PI260429) that were known to harbor the PMMoV-resistant L+ to L4 loci. L4SC340 showed a band for L+, L2, L3 and L4, but not for plants harboring the L1 locus (Fig. 5). In ECW, the band was weaker than in the other plants having the L locus. This result suggests that L4SC340 can detect all pathotypes among the L loci in PMMoV-resistant plants except L1.

Discussion Genetic studies of tobamovirus resistance are well developed in pepper plants (Boukema, 1980). Resistance can be easily screened by eye due to its simple outcome, the presence or absence of necrotic lesions on the inoculated leaf of the tested plant. Although the introgression of tobamovirus resistance into elite breeding lines of comer-

A

B

C

D

E

Fig. 4. PCR analysis of four markers linked to the L locus, and segregation analysis of L4SC340 in F2 individuals. Preparation of the R and S bulks is described in Materials and Methods. L4SC340 (A) was developed in this study, PMFR11 (B) and PMFR21 (C) were developed by Matsunaga et al. (2003) for L3. WA31-1500S (D) was reported by Sugita et al. (2004) for L4. A total of 216 individuals (104 T101 and 112 T102) were tested with L4SC340 (E). The results indicated segregation of the markers in twenty individuals per population.

cial F1 pepper seeds has been achieved by a traditional breeding program, breeding for virus resistance could be expedited by the development of molecular markers for the L locus. In this study we developed a SCAR marker by converting an AFLP marker closely linked to the L4 locus. This new SCAR marker is situated closer to the L4 allele than a previously reported marker, WA31-1500S (Matsunaga et al., 2003). Our genetic populations consisted of more than 120 individuals each, whereas the F 2 population for WA31-1500S was based upon 65 individuals. WA31-

Hyun Jung Kim et al.

Fig. 5. Agarose gel analysis of PCR bands with primer pairs of the L4SC340 marker on pepper differentials; Capsicum annuum EarlyCalwonder (ECW), C. annuum Tisana (TIS), C. frutescense Tabasco (TAB), C. chinance PI159236 (CHI), C. chacoense PI260429 (CHA). Genotypes for each differential are presented at the top. M is a 1 kb plus ladder molecular marker.

1500S showed monomorphism in our mapping population, indicating the need for sequence extension to obtain new polymorphism. Other markers linked to the L locus have been previously reported: one RFLP, two SCARs, and one RAPD. The RFLP marker TG36 is not practical for tobamovirus resistance screening due to the need to use a radioisotope, and its 4 cM distance from the L locus (Lefebvre et al., 1995). The markers, PMFR11 and PMFR21, developed by a Japanese group for the L3 allele (Matsunaga et al., 2003), could not be used in our mapping population. Our results indicate that the narrowing down of the junction of the L4 allele of wild type peppers such as C. chacoense PI260429, as well as differences in genetic backgrounds, may further affect the applicability of previously reported markers for screening for the L4 allele. Despite this limitation, L4SC340 was useful for screening for the L4 allele in the two elite lines that serve as the parents for commercial F1 pepper seeds and is thus expected to provide a relative advantage in MAS.

Acknowledgments This research was supported by a grant from SeminisKorea and the Center for Plant Molecular Genetics and Breeding Research (CPMGBR) through the Korea Science and Engineering Foundation (KOSEF) and the Ministry of Science and Technology (MOST).

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