Genetic and sequence analysis of markers tightly linked to the soybean mosaic virus resistance gene, Rsv3. (Cell Biology & Molecular Genetics).
The gene, Rsv3, was first identified in `OX686', a line derived from the cultivar Columbia (Tu and Buzzell, 1987). Bowers et al. (1992) found that the soybean line `HLS', derived from the cultivar Hardee, carried a single dominant gene at a locus independent of the Rsv1 resistance gene. Allelism tests involving Columbia and `L29' (an isoline derived from Hardee, Bernard et al., 1991), and lines containing Rsv1 alleles showed that the resistance gene in Columbia and Hardee, which is independent of the Rsv1 gene, most likely is located at the same locus (Buss et al., 1999).
Rsv3 is unlike the well-characterized Rsv1 alleles in terms of the degree of resistance to seven SMV strain groups (G1-G7 classified on the basis of their virulence; Cho and Goodman, 1979). Rsv3 confers resistance to the more virulent strain groups, G5 through G7, and conditions stem-tip necrosis or mosaic symptoms to the less virulent groups, G1 through G4 (Tu and Buzzell, 1987; Buzzell and Tu, 1989; Bowers et al., 1992). By contrast, Rsv1 generally confers resistance to the less virulent (lower numbered) strain groups and conditions necrotic or mosaic reactions to more virulent (higher numbered) groups (Chen et al., 1991).
The use of molecular-marker mapping can facilitate both MAS and map-based cloning of disease resistance genes. The objectives of this study were to locate the Rsv3 gene to a MLG and to map the chromosomal region flanking the gene using molecular markers. Molecular markers, including RFLP, AFLP, microsatellites, and other PCR-based markers, were used to map Rsv3 and two high throughput PCR-based markers were developed for the purpose of MAS.
MATERIALS AND METHODS
Plant Genetic Material
Seeds of L29, a backcross-derived isoline of `Williams' carrying the Rsv3 resistance gene from Hardee, were obtained from R. L. Bernard at the University of Illinois (Urbana-Champaign, IL). A cross was made between L29 and the susceptible cultivar Lee68 (hereafter, LL population). In total, 195 [F.sub.2:3] lines were evaluated from this mapping population. A second population of 149 [F.sub.2] individuals (Maughan et al., 1996) of an interspecific cross between a Glycine max line (rsv3) `V71-370' and a G. soja Siebold & Zucc. line (rsv3) `PI406.162' (hereafter, referred to as the VP population) was used to quickly assign Rsv3 to a previously defined linkage
group. In this latter population, which contains over 400 mapped marker loci (Saghai Maroof, unpublished), all 20 soybean MLGs have been identified. A third [F.sub.2] population of 62 individuals from a cross between `Tousan 140' (Rsv3) and Lee68 (hereafter, TL population) (Gunduz, 2000) was used to confirm the location of Rsv3 and to assign additional markers that were not polymorphic in the LL population.
SMV Disease Reaction
The virus-reaction phenotype of each [F.sub.2] plant from the LL and TL populations was determined by inoculating [F.sub.2:3] plants with SMV strain G7 (supplied by S.A. Tolin at Virginia Tech) in the greenhouse. Twenty seeds from each [F.sub.2:3] line were planted in 15-cm plastic pots containing a 1:1 mixture of top soil and commercial potting soil. A set of six SMV strain differentials, `PI96983', `York', `Ogden', `Marshall', Lee68, and L29 were included as checks in the experiment. The inoculum was maintained on the susceptible cultivar York. Inoculations were performed approximately 10 d after planting when the unifoliolate leaves were fully expanded (Hunst and Tolin et al., 1982). Plants were scored for disease reaction at 2 and 4 wk after inoculation. Reactions were recorded as either resistant (symptomless) or susceptible (mosaic).
Soybean leaf tissue was used for DNA extraction. Soybean DNA samples were prepared from freeze-dried tissue as described previously (Saghai Maroof et al., 1984). Parental lines L29 and Lee68, as well as Williams and Hardee, the recurrent and donor parents for L29, respectively, were screened with molecular markers to detect polymorphism. Resistant and susceptible pools for bulked segregant analysis (Michelmore et al., 1991) consisted of DNA from 15 homozygous resistant and 15 homozygous susceptible [F.sub.2] plants of the LL population.
For RFLP analysis, DNA digestion and hybridization were carried out essentially as described previously (Yu et al., 1994). The restriction enzymes BamH1, DraI, EcoRI, EcoRV, HindIII, BclI, TaqI, and XbaI were employed. Publicly available RFLP probes were kindly provided by R. Shoemaker (Iowa State University/USDA/ARS, Ames, IA). AFLP analysis was carried out following the protocols as described previously (Vos et al., 1995; Maughan et al., 1996). DNA was digested with restriction enzymes EcoRI and MseI followed by ligation with specific adaptors for + 1 and +3 amplification. AFLP products were visualized by means of a [alpha]-[sup.32]P end-labeled Eco primer and electrophoresis through a 7 M urea, 6% (w/v) polyacrylamide gel for 3h at 45 W. AFLP fragments of interest were cloned into a plasmid according to the methods of Upender et al. (1995) and Hayes and Saghai Maroof (2000). In brief, a polymorphic AFLP band eluted from a gel slice was reamplified with specific +3 primers, by cold PCR. Product of the reamplification was resolved on an agarose gel and visualized by ethidium bromide staining to estimate the fragment size. This fragment was then cloned into the pCNTR shuttle vector (5prime-3prime, Boulder, CO) or cloned into the pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA) following the manufacturers' protocols. The plasmid insert was then used as a probe for RFLP analysis.
Amplification by means of microsatellite primer sets was carried out as described by Yu et al. (1994) and Cregan et al. (1999). PCR products were resolved by means of a 6.5% polyacrylamide gel and visualized as described previously (Saghai Maroof et al., 1994). Publicly available microsatellite primer sequences were kindly provided by P. Cregan (USDA/ ARS, Beltsville, MD). The primers were obtained from Research Genetics Inc. (Huntsville, AL) or custom made by Gibco-BRL Life Technologies (Rockville, MD).
Genomic Library Screening
A genomic library of the soybean line `L81-4420' (a Williams isoline) was custom made by Clontech (Palo Alto, CA) using the EMBL SP6/T7 vector. This library was screened with an RFLP marker Mng247 (provided by N. Young, University of Minnesota, St. Paul, MN) according to the manufacturer's protocol. Mng247 is a mungbean clone that identifies one locus each on linkage groups B2 and G as specified on the USDA Soybase composite maps (http://macgrant.agron. iastate.edu/; verified August 8, 2001). Inserts of positive lambda clones were digested with SstI and subcloned into pBluescript II KS(-) plasmid (Stratagene, La Jolla, CA) according to the manufacturer's protocol.
PCR products were prepared for sequencing by excising a band of expected size from an agarose gel followed by purification by QiaexII (Qiagen, Valencia, CA). When necessary, a given PCR product was subcloned into a plasmid for sequencing. Plasmid templates were prepared using standard alkalinelysis and then purified using QiaexII. Dye-terminator chemistry was performed according to the manufacturer's protocols (Perkin Elmer, Foster City, CA) and sequences were visualized by means of an ABI377 DNA Sequencer (Perkin Elmer). Sequence analysis including primer design was performed by Lasergene software (DNASTAR, Madison, WI).
Segregation ratios for SMV disease reaction and molecular marker data from screening the [F.sub.2] population were tested for goodness of fit to a 1:2:1 genotypic ratio by Linkage-1, a Pascal computer program developed by Suiter et al. (1983). Linkage analysis was performed by MapMaker 3.0b (Lander et al., 1987) at log likelihood 3.0, with a maximum Haldane distance of 50 centimorgan (cM). To verify the order of markers obtained by three-point analysis, the Ripple command was used at window-size 5 and log-likelihood threshold 3.0. No significant alternative orders were revealed by MapMaker in the analyses of either the LL or TL populations. The Kosambi function was used to calculate map distances with error detection on.
Linkage Mapping of Rsv3
Disease reaction of [F.sub.2:3] families from the LL population was assessed following inoculation with SMV strain G7. The segregation for resistance to SMV displayed a 1:2:1 ratio (homozygous resistant: heterozygous: homozygous susceptible, [chi square] = 2.77, P = 0.25). AFLP marker analysis of parental lines and bulk segregants of the LL population, as well as Williams and Hardee, which are ancestral lines of L29, identified DNA fragments putatively linked to Rsv3. An AFLP fragment of approximately 80 base pairs (bp) was detected with primer combination Eco + AAC/Mse+ CTG. This AFLP fragment was converted to an RFLP probe named ACR1. Linkage of ACR1 to Rsv3 was confirmed by RFLP mapping in the LL population (Fig. 1A). ACR1 was mapped to MLG B2, a previously defined linkage group, between the RFLP markers A516 and B221 in the VP population (data not shown). An additional AFLP fragment of approximately 340 bp putatively linked to Rsv3 was detected with the primer combination Eco +AAG/ Mse+CGA. This AFLP fragment was also converted to an RFLP marker named ACR2, and then mapped in the LL population (Fig. 1A).
[FIGURE 1 OMITTED]
The results obtained from AFLP analysis facilitated identification and placement of molecular markers closely linked to the chromosomal region flanking the Rsv3 gene. In total, we mapped three RFLP markers (A519, A516, and A593), two AFLP converted to RFLP markers (ACR1 and ACR2), and two microsatellite markers (Satt63 and Satt534) to one side of the vicinity of the Rsv3 gene (Fig. 1A). A519, which maps 0.9 cM away from Rsv3, is the locus closest to Rsv3. However, we were unable to map any marker to the other side of the Rsv3 gene in this population.
PCR-Based Markers Tightly Linked to Rsv3
The RFLP clone, A519, was converted to a PCR-based marker for future use in MAS. DNA fragments corresponding to RFLP clone A519 were PCR-amplified using primers A519-5' and A519-3' (Coryell et al., 1999) from DNA of L29 and Lee68. PCR fragments were cloned and sequenced. The A519 sequence of L29 (GenBank accession no. AF348331) has two single-base substitutions and one 4-base indel (insertion/deletion) relative to that of Lee68 (GenBank accession no. AF348332) (Fig. 2A). The indel site corresponds to the recognition site for the AseI restriction enzyme. Using this sequence information, we designed specific forward and reverse primers in order to PCR-amplify the DNA region spanning the 4-base indel. The primer set generated a codominant PCR marker in the LL population and was named A519F/R. A519F/R cosegregates with RFLP marker A519.
[FIGURE 2 OMITTED]
Mng247, a mungbean clone that detects loci on the soybean linkage groups B2 and G, is expected to map very close to Rsv3 based on comparative positioning of the clone on the VP map and the map of Cregan et al. (1999) (data not shown). However, Mng247 is not polymorphic in the LL population. Sequencing and subsequent sequence analysis of the Mng247 insert indicate that approximately 200 bp from one end of this clone (total size of 1800 bp) is similar to the C-terminal region of plant protein kinases. To identify soybean genomic clones corresponding to Mng247, this clone was used as a probe to screen a soybean genomic library of the line L81-4420. Two genomic clones, M1 and M3, were analyzed on the basis of their RFLP patterns. An SstI digested subclone of M1 (3.0 kb, hereafter referred to as M1a) shows an RFLP pattern similar to that of Mng247 when hybridized with soybean parental DNA on a Southern blot. An SstI digested subclone of M3 (3.5 kb, hereafter referred to as M3a) shows a simple RFLP pattern that is different than the pattern seen with the original Mng247 probe.
Both ends of M1a and M3a were sequenced. None of these sequences showed significant similarity with Mng247 insert sequence. One end sequence of M1a (GenBank accession no. AF348333) shows a putative N-terminal region as well as part of an LRR similar to that of the extracellular LRR superfamily of resistance genes (Fig. 3). The consensus sequence LXXLXXLXXLXLXXNXLXGXIPXX of the M1a LRR is identical to that of the extracellular LRR resistance genes, Cf9 and Xa21 (Jones et al., 1994; Song et al., 1995). One end sequence of M3a (GenBank accession no. AF348334) contains three different microsatellite repeats spanning 200 bp (Fig. 2B). The microsatellite repeats were exploited by designing the forward and reverse primers (hereafter referred to as M3Satt) for PCR amplification.
[FIGURE 3 OMITTED]
Confirmation of Linkage Relationship of Rsv3
The genomic location of Rsv3 was confirmed in the TL [F.sub.2] mapping population. This was necessary, because all of our mapped markers were on one side of Rsv3 in the LL population. Three PCR markers and seven RFLP markers were mapped in this second population (Fig. 1B). It is noteworthy that the Mng247-derived soybean markers, M1a, M3a, and M3Satt, which are monomorphic between L29 and Lee68, map 0.8 cM away from Rsv3 on the distal side where no marker was located in the LL population. These three marker loci cosegregate with each other and are separated from Rsv3 by one recombination out of 62 [F.sub.2] individuals tested. A519 cosegregates with Rsv3 in this population. A519F/R is monomorphic in the TL population. When visually comparing the maps generated by the LL and TL populations, marker order is identical for both maps and only slight differences in genetic distances are observed (Fig. 1A and 1B).
We have mapped the Rsv3 gene, conferring resistance to Soybean mosaic virus, between markers A519 and M3a of MLG B2. This study completes the molecular mapping efforts of the three reported independent SMV resistance genes, Rsv1, Rsv3, and Rsv4. Rsv1 has been mapped to MLG F (Yu et al., 1994) and Rsv4 to MLG D1b (Hayes et al., 2000). The culmination of this work now enables us to use molecular markers to combine different sources of SMV resistance into a single elite line or cultivar with the objective of achieving durable resistance. The Rsv3 gene is particularly interesting in the efforts for pyramiding SMV resistance genes, because it confers resistance to the more virulent strain groups, G5 to G7. We have converted two RFLP markers closely linked to Rsv3 to the PCR-based markers A519F/R and M3Satt. In soybean, RFLP markers frequently map to two or more loci reflecting its ancient tetraploid nature (Shoemaker et al., 1992). However, PCR markers, including microsatellite markers, map to one locus in most cases (Cregan et al., 1999). Only a few PCR-based markers, including Satt63, Satt534, and Satt560, have been reported in this region of chromosome B2. Satt560 is monomorphic in the LL and TL mapping populations. Thus, the two additional high throughput PCR-based markers A519F/R and M3Satt identified in this study, should allow us to speed up MAS of Rsv3-carrying lines as well as pyramid the three independent SMV resistance genes.
In addition to their immediate utility for marker assisted selection, the molecular markers reported in this study will be useful for map-based cloning of Rsv3 in the future. We developed two high throughput PCR-based markers linked to the Rsv3 gene. Furthermore, one of the newly identified RFLP markers contains an open reading frame (ORF) coding for a putative LRR sequence. This sequence is similar to a major superfamily of disease resistance genes that encodes an extracellular LRR (for a review, see Ellis and Jones, 1998). In recent years, several disease resistance genes have been cloned by positioning the targeted resistance locus by means of molecular markers, some of which contain the conserved motifs of the cloned resistance genes (e.g., Song et al., 1995; Meyers et al., 1998).
The chromosomal region in the proximity of Rsv3 appears to contain a cluster of disease resistance genes. In this chromosomal region, significant quantitative associations with resistance to two races of soybean cyst nematode have been reported (Qiu et al., 1999). This region is also the putative location of Rps5 conferring resistance to Phytophthora sojae M.J. Kaufman & J.W. Gerdemann (Diers et al., 1992). The clustering of disease resistance genes has been reported in many plants (for a review, see Michelmore and Meyers, 1998). In soybean, clusters of several closely linked resistance genes have been reported on MLG J and MLG F (e.g., Polzin et al., 1994; Ashfield et al., 1998). These resistance gene clusters have been shown to be associated with gene candidates that are similar to previously cloned disease resistance genes (Yu et al., 1996; Kanazin et al., 1996). Rsv1 maps to the cluster of disease resistance genes on MLG F and is tightly linked to several gene candidates belonging to the nucleotide binding site/ LRR class of disease resistance genes (Jeong et al., 2001). The genomic clone, M1a, tightly linked to the Rsv3 gene, contains an LRR consensus region identical to that of the extracellular LRR class of disease resistance genes. Given the unique specificities associated with these two SMV resistance genes it is interesting to note that they are positionally associated with different classes of disease resistance candidates. These observations may provide insights toward the eventual cloning of these important virus resistance genes.
This study was supported in part by the USDA NRICGP Grant no. 96-35300-3648 and by the United Soybean Board.
Abbreviations: AFLP, amplified fragment length polymorphism; bp, base pairs; cM, centimorgan; LRR, leucine-rich repeat; MAS, marker-assisted selection; MLG, molecular linkage group; NIL, near isogenic line; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; SMV, Soybean mosaic virus.
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S. C. Jeong, S. Kristipati, A. J. Hayes, P. J. Maughan, S. L. Noffsinger, I. Gunduz, G. R. Buss, and M. A. Saghai Maroof *
S.C. Jeong, S. Kristipati, A.J. Hayes, I. Gunduz, G.R. Buss, and M.A. Saghai Maroof, Crop and Soil Environmental Sciences, Virginia Tech, Blacksburg, VA 24061-0404; P.J. Maughan, Monsanto Company, 3302 SE Convenience Blvd., Ankeny, IA 50021; S.L. Noffsinger, USDA-ARS Small Fruits Research Unit, 306 S. High St., Poplarville, MS 39470. Received 16 Feb. 2001. * Corresponding author (smaroof@ vt.edu).