Evaluation of PCR-based markers for scanning tomato chromosomes for introgressions

from wild species

 

Christopher T. Martin1, Melinda S. Salus1, Brenda E. Garcia1,2, Katie S. Jensen1, Luis Montes1,2, Carolina Zea1,2, Sergio Melgar1,2, Khadija El Mehrach1,3, Julieta Ortiz1,2, Amilcar Sanchez1,2, Michael J. Havey4, Luis Mejía1,2, and Douglas P. Maxwell1

 

1Department of Plant Pathology, University of Wisconsin-Madison, Madison, WI 53706

2Universidad de San Carlos, Guatemala City, Guatemala

3Université Ibn Zohr, Agadir 80000, Morocco

4Department of Horticulture, USDA, University of Wisconsin-Madison, WI 53706

            Email: dpmax@plantpath.wisc.edu

 

Introduction:  Marker-assisted breeding can increase the efficiency of introducing disease resistance into tomato breeding lines (Foolad and Sharma, 2005; Scott, 2005).  Excellent chromosome maps of markers are available on the SGN web site (www.sgn.cornell.edu), and these genetic maps have RFLP, SSRs, BAC clones, cDNAs, CAPS, ESTs, and COSII markers.  The objective of our project was to locate introgressions associated with begomovirus-resistance genes in germplasm from the tomato breeding program at San Carlos University, Guatemala (Mejía et al., 2005).  PCR fragments were amplified using primers for markers from the SGN web site, if available, and by designing primers, if not.  These PCR fragments were directly sequenced, and the sequence of fragments from begomovirus-resistant inbred lines were compared with each other and the corresponding fragments from susceptible lines.  The presence of SNPs or indels in fragments from resistant-inbred lines was assumed to indicate an introgression from a wild species.

 

Germplasm tested:  Begomovirus-resistant inbred lines used in this study were part of the tomato breeding program at San Carlos University, Guatemala City (Mejía et al., 2005).  The original sources of begomovirus-resistance were hybrids or lines generously provided by colleagues from public institutions.  Inbred lines derived from the hybrids (e.g., FAVI-9) provided by F. Vidavsky and H. Czosnek (1988), which had resistance genes introgressed from Solanum habrochaites LA1777 and/or LA0386, are designated G for Guatemala, then h for Hebrew University of Jerusalem (HUJ), and numbers for the cross and the selection, e.g., Gh25-3.  The resistance genes in these lines originated from line 902 (Vidavsky and Czosnek, 1988).  No introgressions had been detected in line 902 at the time (1998) the germplasm was received in Guatemala.  P. Hanson, Asian Vegetable Research and Development Center, Taiwan, provided inbred line, H24, with resistance derived from the Ty-2 locus of S. habrochaites.  This line was used solely as the standard for the introgression in chromosome 11 (Hanson et al., 2000, 2006).

Three sources of begomovirus-resistance from S. chilense were obtained.  J.W. Scott, University of Florida, provided begomovirus-resistant inbreds, Fla. 595-2 and Fla. 8348, derived from S. chilense LA2779 and LA2779/LA1932, respectively.  These Florida inbreds have introgressions on chromosome 6 (Agrama and Scott, 2005; Ji and Scott, 2005, 2006; Ji et al., 2007).  The lines Gc9 and Gc171 were selected from Fla. 595-2 and Fla. 8348, respectively.  Gc9 was crossed with susceptible hybrids, and resistant-inbred lines were derived from these populations.  These selections were designated G for Guatemala, then c for S. chilense, and numbers for the cross and the selection, e.g. Gc143-2.  H. Czosnek, HUJ, provided TY52, an inbred with resistance from S. chilense LA1969.  This line was used as the standard for the introgression for the Ty-1 locus on chromosome 6 (Zamir et al., 1994).

            Susceptible germplasm included M82-1-8 (Ve, F1, HUJ), Heinz 1706 (Ve, F1, Heinz Seeds), HUJ-VF (Ve, F1, HUJ), the heritage tomatoes, Purple Russian and German Pink, and the landrace of S. lycopersicum var. cerasiformae (cera), which was collected in Sanarate, Guatemala.

 

Results and Discussion:  Primer information, sequence data and marker detection protocols for this report are available at the web site:

            www.plantpath.wisc.edu/GeminivirusResistantTomatoes/markers.html

For several chromosomes, resistance-gene hot spots were selected as the most likely regions for detecting introgressions (Pan et al., 1999).  Eighty-eight markers were evaluated on eight chromosomes.  One, nine, two, one, one, and seven markers were amplified and sequenced from chromosomes 1, 2, 4, 7, 9, and 12, respectively.  No evidence for introgressions from wild species was detected in begomovirus-resistant germplasm for these markers.  For chromosomes 6 and 11, sequence data are reported for 38 and 15 markers, respectively, and introgressions at several markers were detected in the begomovirus-resistant germplasm.

Chromosome 6, which is known to have various resistance genes (Agrama and Scott, 2006; Foolad and Shrama, 2005; Zamir et al., 1994), was studied most extensively.  Markers at 5-cM intervals were amplified and sequenced.  Gc9 (LA2779) and lines derived from Gc9 had an introgression from LE_HBa0037I09 (5 cM) to T0834 (32 cM).  No introgression was detected from the top of the chromosome to T0270 (3.0 cM) or from marker TG365 (33.5 cM) to the bottom of the chromosome 6.  Our results support the recent report by Ji et al. (2007) that some LA2779-derived inbreds similar to Gc9 had an introgression from 5.3 to 32 cM, which included the Ty-1 and Ty-3 regions.  To determine if Gc9 had the Ty-1 locus, the sequence for the introgression at the Ty-1 locus (TG97, 8.5 cM, Zamir et al., 1994) of Gc9 was found to be identical to that for this region of line TY52 (Ty-1/Ty-1), and a CAPS marker was developed for this locus (Protocol I for Ty-1 locus).  A SCAR marker (P6-6-F1/P6-6-R1 primers) was found at 5.5 cM for the S. chilense introgression in TY52 and some Gc9-derived lines, but this marker is not tightly linked to the Ty-1 gene (Protocol II for the Ty-1 locus).  For the Ty-3 locus, FLUW25 and P6-25 (25 cM), co-dominant SCAR markers, were developed.  Surprisingly, sequences of 17 markers from 6 cM (Mi23) to 32 cM (T0834) from lines derived from 902, which was derived from S. habrochaites (Vidavski and Czosnek, 1998), were identical to those from Gc9.  This region included the loci for the Ty-1 and Ty-3 introgressions.  One difference between 902-derived lines and Gc9 was that the REX-1 marker (ca. 5 cM) from 902-derived lines had sequences identical to that of the REX-1 marker from lines with the Mi-1.2 gene (introgressed from S. peruvianum) for resistance to root-knot nematode.  Conversely, the sequence for the REX-1 marker for Gc9 was identical to that of TY52 (sequence from S. chilense).  A co-dominant SCAR marker (Mi23, ca. 6 cM) for the Mi-1.2 gene was evaluated using primers developed by S. Seah and V. Williamson (per. com., University of California-Davis).  This Mi23 SCAR marker is tightly linked to the Mi-1.2 gene and can be used as a CAPS marker for the Ty-1 locus in some lines (Protocol III for the Ty-1 locus).  In addition, four sets of primers were designed from the REX-1 marker sequences from various wild species, and these could be used as internal control standards (ICON primers) in multiplex PCR.

From this study of the markers for chromosome 6 with Gc9 and 902-derived lines, it is proposed that the order of markers in these lines is REX-1, Mi-1.2, acid phosphatase, Ty-1 and Ty-3.  This order is supported by the observation that 902 has the REX-1 marker, but lacks the Mi-1.2 gene.  Lines with only the Ty-1 marker (TG97) or the Ty-3 marker (FLUW25) were detected in 902-derived lines, and one line, Gh2, was homozygous for Rex-1, Mi23, Ty-1 (TG97) and Ty-3 (FLUW25) markers.  As expected from the marker analysis, this line, Gh2, was resistant to root-knot nematode (bioassay by V. Williamson, Univ. of California-Davis) and also resistant to begomoviruses.

Chromosome 11 is known to have an introgression from S. habrochaites for the Ty-2 gene between TG36 (84 cM) and TG26 (92 cM) (Hanson et al., 2000, 2006).  Primers for four markers for this region were evaluated.  Primers (T0302F/Ty-2R2) for the T0302 marker amplified different size fragments for susceptible and resistant genotypes (co-dominant T0302-Ty-2 SCAR Marker).  Since the I-2 gene (91.5 cM) for resistance to Fusarium oxysporum f. sp. lycopersicum race 2 is closely linked to the Ty-2 locus, it was a concern that the T0302-Ty-2 SCAR marker might give a false positive with I-2/I-2 genotypes.  Fortunately, no alien introgression was detected in lines that had the I-2 locus and lacked the Ty-2 locus.  No lines with the Ty-2 and I-2 loci were available to test.

            For many markers, sequence is provided on the web site for various accessions of S. chilense, S. peruvianum, and S. habrochaites, e.g. REX-1, Mi23, FLUW25, T0834, and T0302.

 

Acknowledgements:  This project was funded in part by USAID-CDR (TA-MOU-05-C25-037) and USAID-MERC (GEG—G-00-02-00003-00) grants to D. P. Maxwell and by the College of Agricultural and Life Sciences at University of Wisconsin-Madison.  Authors thank colleagues who so generously provided germplasm and DNA samples.  D.P. Maxwell and L. Mejía especially thank Dr. Uri Lavi, Volcani Center, Israel, for introducing us to SNPs and indels.

 

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