El Mehrach, K., Ibn Zohr University, Agadir, Morocco; S. Gharsallah Chouchane, University of Wisconsin-Madison; L. Mejía, Universidad de San Carlos, Guatemala; V. M. Williamson, University of California-Davis; F. Vidavski, The Hebrew University of Jerusalem; A. Hatimi and Saida Tahrouch, Ibn Zohr University; M.S. Salus, C.T. Martin, and D.P. Maxwell, University of Wisconsin-Madison.
Abstract
The begomovirus-resistant breeding lines being developed in Guatemala were tested for the CAPS markers, REX-1 and Cor-Mi, that are linked to the Mi-1 locus for root-knot nematode resistance. Both markers gave false positive results for several breeding lines that are susceptible to M. incognita and have L. hirsutum (line Ih902) or L. chilense (line TY52) introgressions. SNPs and indels specific to introgressions from two wild species, L. peruvianum and L. chilense, are described for the two CAPS markers. A multiplex PCR was developed, which involved the REX primers and PCR primers (PM3Fb/PM3Rb) specific for the region 3’ of the Mi-1.2 gene. Every plant DNA tested gave a 720-bp fragment for the REX primers, but only those plants with the Mi-1.2 gene gave a 500-bp fragment with the PM3Fb/PM3Rb primers. Since this multiplex PCR could not detect heterozygous genotypes for the Mi-1.2 gene, another PCR method was used with primers (PMiF3/PMiR3), which gave unique fragment sizes for the Le-Mi-1 locus, the Lp-Mi-1 locus and the Lc-Mi-1 locus, as well as with the begomovirus-resistant line with the Ih902-Mi-1-locus. These two PCR protocols will have application in breeding programs in which parental lines have given false positives with the REX-1 and Cor-Mi markers, i.e., especially those using parental lines with begomovirus-resistance genes introgressed into chromosome 6S.
INTRODUCTION
Resistance to root-knot nematode (RKN, Meloidogyne spp.) in Lycopersicon esculentum is controlled by a single dominant gene, Mi-1 at the Mi-1 locus, which was introgressed from the wild species L. peruvianum (Smith, 1944). The Mi-1 locus is linked to RFLP markers on chromosome 6S (Ho et al., 1992); and the functional gene, Mi-1.2, has been sequenced (Milligan et al., 1998). Recently, Seah et al. (2004) reported that there are seven homologs of the Mi gene arranged in clusters of three (cluster 1p) and four (cluster 2p) homologs in the resistant cultivar Motelle [L. peruvianum (Lp) introgression]. The Mi-1.2 gene is in cluster 1p. In the susceptible cultivar Moneymaker [L. esculentum (Le)] a similar arrangement of the seven homologs (cluster 1e and cluster 2e) exists. One difference between the organization of the Mi-1 locus in Lp and Le is that the 300-kb region between the two clusters is inverted in Le. Two additional homologs in Le were mapped to chromosome 5. Other genes for disease resistance have been mapped to the chromosome 6S (see Pan et al., 2000; Zhang et al., 2002), e.g., Ty-1 gene for resistance to Tomato yellow leaf curl virus in line TY52 (Zamir et al., 1994).
Marker-assisted selection offers the advantage of tagging resistance genes without biological assays. Two codominant CAPS (Cleavage Amplified Polymorphic Sequence) markers for detection of the Mi-1 locus have been developed. Williamson et al. (1994) reported the REX-1 marker (PCR primers, REX-F1/REX-R2); and Tanksley et al. (contact Cornell Univ. Foundation, Ithaca, NY) developed another, the Cor-Mi marker (PCR primers, Cor-F1/Cor-R2).
The objectives of this research were: i) to evaluate the REX and Cor-Mi CAPS markers for tagging the Mi-1 locus in begomovirus-resistant tomato germplasm that had introgressions for begomovirus resistance from L. chilense (Lc, Zamir et al., 1994) or L. hirsutum (Vidavsky and Czosnek, 1998), and ii) to develop alternative PCR-based methods for tagging the Mi-1 locus, if necessary. The begomovirus-resistant germplasm is currently being evaluated for resistance to begomoviruses in Morocco and Guatemala (Mejía et al., 2004); and the expectation is that these PCR-based methods for tagging the Mi-1 locus will allow the development of breeding lines with resistance both to begomoviruses and root-knot nematode.
MATERIALS AND METHODS
The known RKN-resistant cultivars with introgressions of the Mi-1 locus were Motelle and Anahu; and susceptible cultivars were Moneymaker, M82, and TY50 (supplied by H. Czosnek). Commercial cultivars designated N for RKN resistance were Better Boy (V1, F1, N, ASC, St.), Marina (V1, F1, F2, N, ASC, St., BS race 0), Sheriff (V, F1, F2, N) and Dominique (V, F1, F2, TMV, N). Begomovirus-resistant germplasm with introgressions from L. chilense were TY52 (Zamir et al., 1994; supplied by H. Czosnek) and Gc9 (Mejía et al., 2004). Also, begomovirus-resistant germplasm with introgressions from L. hirsutum were Ih902 (listed as 902 in Vidavsky and Czosnek, 1998), and Gh13 and Gh2 (breeding lines selected in Guatemala from a hybrid of Ih902 by begomovirus-susceptible lines, Mejía et al., 2004).
Root-knot nematode bioassays with Meloidogyne incognita were performed at the University of California-Davis and at The Hebrew University of Jerusalem using standard protocols.
DNA was extracted from fresh leaves from plants grown in a plant growth chamber at the University of Wisconsin-Madison. Thirty mg of tissue was frozen in liquid nitrogen in a microfuge tube, then ground with a sterilized Kontes™ micropestle (Kontes Glass, Vineland, NJ), and extracted with the PUREGENE® DNA Purification Kit (Gentra Systems, Inc., Minneapolis, MN) following the manufacturer’s instructions. DNA concentrations were adjusted to 10 ng/µl and extracts frozen at -20oC. All PCR primers were purchased from Integrated DNA Technologies, Inc., Coralville, IA. PCR parameters were for 50-µl reactions containing 5 µl 2.5 mM deoxynucleotide triphosphates (dNTPs), 5 µl 10x buffer, 5 µl 25 mM MgCl2, 0.2 µl Taq DNA polymerase, 5 µl each forward and reverse sense primer at 10 µM, 5-7 µl of DNA extract, and H20. PCR cycle parameters for fragment amplification were as follows: denaturation at 94°C for 3 min, then 35 cycles 94°C for 30 sec, annealing at 50 or 53°C for 1 min, and extension at 72°C for 1 min. These cycles were followed by a reaction at 72°C for 10 min, and then the reaction was held at 4°C. PCR reactions were performed in the MJ DNA Engine PT200 Thermocycler™ (MJ Research Inc., Waltham, MA). All molecular biology chemicals for PCR were purchased from Promega, Corp., Madison, WI. PCR-amplified fragments were separated by gel electrophoresis using 1.5% Seakem LE™ agarose (BioWhittaker Molecular Applications Rockland, ME) in 0.5X TBE buffer, stained with ethidium bromide, and visualized with a Kodak Gel Logic 200 Imaging System. PCR fragments were directly sequenced using Big Dye Sequencing Kit™ (Biotechnology Center, Madison, WI). Analysis of the sample sequences was accomplished by comparison with known DNA sequences through the National Center for Biotechnology Information BLAST program and the DNAMAN software (Lynnon Corp., Quebec, Canada).
RESULTS AND DISCUSSION
Tagging the Mi-1 locus in control cultivars
As expected, the same size PCR fragment (720 bp) was obtained with the REX primers for Motelle, Anahu, Moneymaker, Better Boy, Sheriff, Dominique, TY52, Gh2, Gh13 and Ih902. Rather than digest these fragments with the TaqI restriction nuclease to detect the CAPS marker, each PCR fragment was sequenced to detect the single nucleotide polymorphism (SNP) associated with the TaqI restriction site (TCGA) for Lp and to determine if there were other sequence differences. The SNP (A/C) associated with the REX-1 marker for Le is nt C. The PCR fragment for the resistance REX-1 marker will digest with TaqI restriction nuclease (SNP A) to give fragments of 570 and 160 bp, whereas there is no TaqI site (SNP C) in the PCR fragment from Le. The SNP nucleotide (nt) A for the Lp introgression was present in the PCR fragment from Motelle (AY589502), Anahu, TY52, Gh2, and Ih902; and the SNP nt C was present at this position in the susceptible cultivars, Moneymaker, Gh13, TY50, and M82 (AY596779). The Gh2 line was resistant to M. incognita and also to the bipartite begomovirus complex in Guatemala. Since the begomovirus-resistant lines TY52 and Ih902 were found to be susceptible to M. incognita, the SNP nt A associated with the TaqI site of the REX-1 marker gave false positives for these two lines.
The sequences of the REX fragments from the cultivars and lines were analyzed for the presence of additional SNPs. Each REX fragment was directly sequenced using the REXR2 primer and about 600 nt were obtained. In this 600-nt region the RKN-susceptible lines, Gh13, TY50, M82, and Moneymaker, had identical sequences, and the three RKN-resistant lines, Motelle, Anahu, and Gh2, had different but identical sequences. Ten SNPs between the susceptible line M82 (AY596779) and resistant cultivar Motelle (AY589502), A/C, G/T, A/G, A/G, A/G, A/C, T/A, A/G, C/T, and C/A, were identified at nt 18, 33, 63, 100, 138, 235, 279, 489, 522, and 549, respectively. SNP positions are based on the nt number for REX sequence for M82. The RKN-susceptible breeding line Ih902 had the same sequence for this region as Motelle, so there is a peruvianum-like sequence in the REX-1 locus in Ih902. The sequence for TY52, which has an introgression from L. chilense in the REX-1 locus region (Zamir et al., 1994), had two TaqI restriction sites, which could be used to distinguish it from the Lp-REX-1 region that only has one TaqI restriction site. When compared with M82, TY52 and Motelle had the same SNPs at nt 33, 100, 138, 235, 489 and 549, and TY52 had unique SNPs, T/C, A/C and C/G, at nt 145, 319, and 559, respectively. Thus, three types of sequences could be recognized by the unique SNPs for the REX-1 locus, i.e. Le-REX-1 (M82), Lp-Rex-1 (Motelle), and Lc-REX-1 (TY52).
It was suspected that the commercial hybrids, Better Boy, Marina, and Dominique, would be heterozygous for the Mi-1 gene (Mi/mi). Sequence analysis of the REX-PCR fragment indicated that all ten SNPs that are associated with differences between Lp and Le for the REX-1 locus were present. Thus, these three cultivars are heterozygous for the Mi gene (Mi/mi) and have the SNPs associated with the Lp- and Le-REX-1 locus for chromosome 6S.
Since the begomovirus-resistant line Ih902 is a main source of resistance for the begomovirus-resistant breeding programs in Guatemala and Morocco, the REX-1 marker, which gives a false positive, could not be used in a marker-assisted selection program. A second CAPS marker, the Cor-Mi, was then tested. Since the information about this marker is protected by an intellectual property agreement with the Cornell University Foundation, Ithaca, NY, only general results are provided (data not shown). This CAPS marker also gave the expected results with the RKN-susceptible cultivars Moneymaker and M82 and with the RKN-resistant cultivars, Motelle and Anahu. However, false positive results were obtained with RKN-susceptible lines TY52 and Ih902, i.e., these two lines had the same CAPS marker as Motelle, and thus, they should be resistant to RKN.
Development of PCR-based methods for tagging the Mi-1 locus
Since both the REX-1 and Cor-Mi CAPS markers gave false positive results with the begomovirus-resistant breeding lines TY52 (Ty-1 gene) and Ih902, several PCR primers were designed by comparing the sequence of the promoter region for the Mi-1.2 gene (mRNA sequence, AF03982, Milligan et al., 1998) and the 2,000-bp region 3’ of the Mi-1.2 gene with the sequences of the Mi-1.1 gene (mRNA sequence, AF039681) and those of the genomic DNA for this region (U81378).
One set of primers, PMi12F1 (5’ GCA ATT CTA GAT CTA GCT ATT TGT TGT TC 3’) and PMi12R2 (5’ CCT GCT CGT TTA CCA TTA CTT TTC CAA CC 3’), was designed using the differences between the sequences of the promoters for Mi-1.2 and Mi-1.1 genes (Milligan et al., 1998). PCR with these primers gave single 620-bp and 720-bp fragments with Moneymaker and Motelle, respectively, and two bands (620 and 720 bp) for the RKN resistant, heterozygous cultivars, Better Boy and Marina. The sequence of the 620-bp fragment (AY729669) from Moneymaker corresponded to the cluster 1e, and that of the 720-bp fragment from Motelle (AY729670) to the cluster 2p (Seah and Williamson, unpublished). With TY52, which has the Ty-1 gene from L. chilense in this region (Zamir et al., 1994), the PCR fragment was 1,000 bp. The Ih902 fragment was 720 bp, an indication of the Lp-Mi-1 locus sequence; thus, this primer pair gave false positive results with the Ih902 line. This primer pair could be used in a breeding program where the RKN-resistant line has the Lp-Mi-1 locus and the other breeding line has the Le-Mi-1 locus, since this primer pair will detect the homozygous and heterozygous genotypes for the Mi-1 locus without digestion with TaqI restriction endonuclease. Also, this primer pair can detect a chilense-like introgression in this region as indicated by the results with TY52; and a similar 1,000-bp fragment was obtained with Gc9, a begomovirus-resistant breeding line selected in Guatemala, which had resistance from L. chilense LA2779 (J. W. Scott, pers. com.). The REX-1 locus for line Gc9 had the SNPs for the Lc-REX-1 locus, further evidence of an introgression in this region from L. chilense.
The PCR primer pair, PM3F (5' CCT GTG ATG AGA TTC CTC TTA G 3') and PM3R (5' ACC CTT TGT TGA GCG ACT TTG CAG C 3'), was designed to amplify the 3’ region of the Mi-1.2 gene, and these primers gave a 750-bp fragment for RKN-susceptible breeding line Gh13, which is resistant to the begomoviruses in Guatemala (Mejía et al., 2004), and has the Le-REX-1 locus sequence. This PCR fragment was sequenced (GenBank 657582) and 593 bp compared with the similar region for the Mi-1.2 gene from the genomic sequence (U81378). Since there were several large gaps between these two sequences, a PCR primer pair, PM3Fb (5’ CAC ACA TGA GGT ATG TTC GTA TTA TGG 3’) and PM3Rb (5’ TCA CAG CCT AGC TTT TGA ATC AGT ACC 3’), was designed to matched only the sequence for this region 3’ of the Mi-1.2 gene. These primers should amplify a 500-bp fragment only when the Lp-Mi-1 locus is present. This primer pair gave a 500-bp fragment only with Anahu (Mi/Mi), Dominique (Mi/mi), Sheriff (Mi/mi) and not with Moneymaker (mi/mi), TY52 (Ty-1/Ty-1), and Ih902 (Lp-REX-1 locus/Lp-REX-1 locus). The sequence of the 500-bp fragment for Anahu (AY731505) had 100% nt identity with the region 3’ of Mi-1.2 gene, and the sequences of this fragment for Sheriff and Dominique had 100% nt identity with that of Anahu. Since no PCR fragments were detected in the Le-Mi-1 locus plants, a multiplex PCR was used with the REX primer pair and PM3Fb/PM3Rb. In this case, all plants gave a 720-bp fragment for the REX primer pair, and only the plants with the Lp-Mi-1 locus gave the additional 500-bp fragment (Fig. 1). The PM3Fb/PM3Rb pair did not give false positives with TY52 and Ih902 (no 500-bp fragments), but this primer pair did not distinguish between homozygous and heterozygous genotypes for the Lp-Mi-1 locus, e.g. Dominique and Sheriff.
Since it is essential to distinguish homozygous and heterozygous Mi-1 locus plants in a breeding program, additional PCR primer pairs were evaluated for the region 3’of Mi-1.2 gene and the promoter region of the Mi-1.2 gene. The primer pairs designed from the Le sequence corresponding to this region 3’ of the Mi-1.2 gave the same size PCR fragment for Moneymaker (mi/mi) and Anahu (Mi/Mi). Thus, several PCR primer pairs were designed using the Le sequence (AY729669) for Moneymaker for the promoter region obtained with primers PMi12F1/PM12R2, which corresponded to the cluster 1e. The PCR primer pair, PMiF3 (5’ GGT ATG AGC ATG CTT AAT CAG AGC TCT C 3’) and PMiR3 (5’ CCT ACA AGA AAT TAT TGT GCG TGT GAA TG 3’), gave a single unique 350-bp fragment for Moneymaker, 550-bp fragment for Anahu, and for the heterozygous cultivars, Dominique and Sheriff, two fragments, 350 bp and 550 bp. The sequence of the 350-bp fragment from Moneymaker had 100% nt identity with the L. esculentum sequence, which was used to design the primers. The sequence of the 550-bp fragment from Anahu (AY731506) was subjected to a BLAST search of GenBank, and a 94% nt identity was obtained for a 361-nt region (nt 111 to 472) with the promoter region of the Mi-1.2 gene (U65668). The begomovirus-resistant cultivars, TY52 and Ih902, had unique patterns with this primer pair. TY52 had fragments at 350 bp and 700 bp, and Ih902 had fragments at 550 bp, 700 bp and 800 bp (Fig. 2). This primer pair distinguished among the Lp-Mi-1 locus introgression in Anahu, the introgression in TY52 for L. chilense, and the introgression in Ih902.
Two-step PCR protocol for detecting the Lp-Mi-1 locus in begomovirus-resistant germplasm
Since Ih902 was to be used as a source of begomovirus resistance in crosses with a RKN-resistant (Mi-1.2/Mi-1.2) tomato line for the breeding programs in Morocco and Guatemala, the possibility of using two PCR reactions to evaluate the progeny from this cross was examined. Two heterozygous genotypes for this Mi-l locus were simulated. The heterozygous plant (Le-Mi-1/Ih902-Mi-1) was achieved by mixing equal amounts of DNA from Moneymaker (Le-Mi-1, begomovirus-susceptible, RKN-susceptible) with Ih902 (Ih902-Mi-1, begomovirus-resistant, RKN-susceptible), and for the heterozygous plant (Lp-Mi-1/Ih902-Mi-1), DNA from Anahu (Lp-Mi-1, begomovirus-susceptible, RKN-resistant) was mixed with that from Ih902. The first step was the multiplex PCR with the REX and PM3Fb/PM3Rb primers at annealing temperature of 53oC (Fig. 3). As expected, the unique 500-bp fragment for the Lp-Mi-1 locus was detected only with Anahu and the simulated heterozygous plant with DNA of Anahu and Ih902; no 500-bp fragment was detected with Moneymaker, Ih902, or the simulated plant with DNA of Moneymaker and Ih902. The 720-bp REX fragment was detected with all four plant DNAs. Thus, this multiplex PCR clearly detected the presence of the Lp-Mi-l locus, but could not be used to distinguish between heterozygous and homozygous plants for the Mi-1 locus. The heterozygous plants (Le-Mi-1/Ih902-Mi, Lp-Mi-1/Le-Mi-1, Lp-Mi-1/Ih902-Mi-1) were detected by a second PCR that used the PMiF3/PMiR3 primer pair at an annealing temperature of 53oC, which would detect the unique fragment sizes for Le-Mi-1 locus, Lp-Mi-1 locus and Ih902-Mi-1 locus. The PCR fragment sizes were 350 bp and 550 bp for Moneymaker and Anahu, respectively, and for Ih902, four fragments were detected, 550, 700, 750, and 800 bp. As expected for the Le-Mi-1 by Ih902 simulated plant, the fragment for Moneymaker plus the fragments for Ih902 were evident. For the simulated heterozygous plant (Lp-Mi-1/Ih902-Mi-1), only the Ih902-Mi-1 locus pattern was evident, and this is because the 550 bp for Anahu is obscured by the similar fragment from Ih902. The importance of this second PCR is that it detects those plants that have the three unique PCR-fragment patterns for heterozygous plants, Lp-Mi-1/Le-Mi-1 (RKN-resistant), Lp-Mi-1/Ih902-Mi-1 (RKN-resistant), and Le-Mi-1/Ih902-Mi-1 (RKN-susceptible) (Fig. 2, Fig. 4).
This two-step PCR protocol can be used in a breeding program involving Ih902 (begomovirus resistance) and parents with RKN resistance (Lp-Mi-1 locus), since they will detect the Lp-Mi-l locus and also distinguish among the heterozygous progenies. It is expected that this two-step PCR protocol will also have application in breeding programs in which introgressions in the Mi-1 locus are from wild tomato species, e.g., L. chilense, are used in crosses with RKN-resistant breeding lines. In addition, the PCR primers (PM3Fb/PM3Rb) that are specific for the Lp-Mi-1 locus anneal within 1,000 bp of the Mi-1.2 gene for root-knot nematode resistance; thus, they are tightly linked to the functional resistance gene.
ACKNOWLEDGEMENTS
This research was supported by a MERC/USAID grant no. GEG-G-00-02-00003-00, a CDR/USAID grant no. TA-MOU-01-C21-008, and the College of Agricultural and Life Sciences, University of Wisconsin-Madison. Authors appreciate helpful discussions with Dr. M. J. Havey, USDA and University of Wisconsin-Madison.
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