Objective: To determine if resistance gene(s) to Guatemalan begomoviruses present in breeding lines selected in Guatemala had introgressions (resistance genes) in the region of chromosome 6 for the Ty1 gene and chromosome 11 for the Ty2 gene.
Luis Mejia, and Amilcar Sanchez, San Carlos University; Andreas Pallides, Israel; Douglas Maxwell and Mike J. Havey, University of Wisconsin
BACKGROUND: Several research programs have introduced genes for resistance to begomoviruses from wild tomato species into L. esculentum. As indicated earlier the main breeding lines being used in Guatemala, which have high levels of begomovirus resistance, are those derived from the program of Vidavski and Czosnek (S. habrochaites (L. hirsutum) as resistance source) and those from Scott in Florida (L. chilense as resistance source). Thus, our resistance gene tagging efforts have concentrated on tagging genes from these sources, and mainly the lines with L. hirsutum resistance genes. The molecular tagging approach involved the development of PCR primers from RFLP probes that are located in the region of known introgressions for Ty1 gene from L. chilense in chromosome 6 (Theor. Appl. Genet. 88:141-146) and for Ty2 gene from L. hirsutum in chromosome 11 (J. Amer. Soc. Hort. Sci. 125:15-20).
Application of PCR-based tagging approach: The general approach was to sequence RFLP probes located in the region of the Ty1 or Ty2 gene introgression. These probes were identified from the published papers and the chromosome maps at the Solanum Genome Network site (www.sgn.cornell.edu). These sequences were used to search for matching gene sequences in GenBank and the Arabidopsis thaliana genome data base (mips.gsf.de/projects/plants). PCR primers were designed to match the conserved sequences in the exons between L. esculentum and A. thaliana, so that at least one intron and more, if possible, would be amplified by the PCR primer pair. The PCR fragments were cloned and sequenced or sequenced directly. These sequences were compared with DNA software such as GCG or DNAMAN.
Chromosome 11 introgression for Ty2 gene:
The Ty2 gene from L. hirsutum var. glabratum accession B6013 is located in chromosome 11 (Figure 1) (Hanson et al. J. Amer. Soc. Hort. Sci. 125:15-20). The probes TG105, TG26 (pUC8, PstI, 2,400 bp), TG30 (PUC8, EcoRI, 2,000 bp), TG36 (pUC8, PstI, 3,200 bp) and TG393 (pGEM4z, PstI, 1,200 bp), which map to this region, were obtained and sequenced. The resistant line, H24, homozygous for Ty2 gene was supplied by Peter Hanson from Asian Vegetable Research Development Center. DNA of Solanum habrochaites (L. hirsutum) accessions 1928, and 1353 and S. habrochaites f. glabratum LA 1223 (Amer. J. Botany 88:1888-1902) was supplied by D. Spooner, USDA and Department of Horticulture, University of Wisconsin-Madison. Susceptible lines L. esculentum line was L. esculentum var. cerasiforme and M82.
Design of PCR primers for amplification of fragments from the region of the Ty2-gene introgression involved development of primers for RFLP markers using the sequence of the ends of the markers TG36, TG30, TG105, TG26, CT120, and TG393. From these effort, the primer pair for the sequence of TG105(M13F) had a high nt identity with (95%) with Solanum demissium BAC clone, AC091627, at 50,320..50,486 was most informative and only this data is provided. Primer P105s (5’-ctt cag aat tcc tgt ttt agt cag ttg aac c-3’) and P105c (5’-atg tca cat ttg ttg ctt gga cca tcc-3’) gave a single 450-bp fragment with various genotypes of tomato. These PCR fragments were directly sequenced (Figure 2).
Conclusions: In a region of about 160 nt, the sequence of H24 with the Ty-2 gene introgression from S. habrochaites for ToLCV resistance shares 10 SNPs and one large indel with the three accessions of S. habrochaites. These are unique for the H24 and the three wild species accessions and clearing demonstrate that an introgression for S. habrochaites has occurred in this region of chromosome 11. Since these SNPs and the indel are not present in Ih902, Gh1, and Gh2, which all have begomovirus resistance derived from S. habrochaites, it is concluded that this TG105 locus does not contain the resistance gene for these lines. TY52, TY50, G1h, cerasiforme, and HC7880 have identical sequences in this region and G1h is resistant to begomoviruses in Guatemala and the other three are susceptible in Guatemala and TY52 has the Ty-1 gene introgression in chromosome 6.
The results with the locus TG105 show that this PCR-based tagging method can be used to tag regions of introgressions for wild species.
For the TG105 locus, the PSNP105s/PSNP105c-PCR fragment was sequenced for 11 tomato lines and the three S. habrochaites accessions. For the 11 tomato lines, there were three SNP-indel patterns, the esculentum type, the habrochaites type, and the chilense type. Lines Gh1, HC7880, M82, Gh3 had the esculentum type, lines G2h, Ih902, Gc9, and Gper11 had the chilense type, and lines H24 and CLN2498-68 had the habrochaites type. Thus, the lines derived from Ih902 in Guatemala, existed in only the esculentum- and chilense-type patterns and not the habrochaites type.
Testing of other primer pair for introgressions of the resistance gene
in Ih902 in chromososme 11 in the region of Ty-2 gene.
Aldehyde Oxidase primers (PALO, Chromosome 11): The TG105 sequence had a high identity to the S. demissum BAC (AC091627) and there were several gene sequences associated with this BAC clone. One gene, aldehyde oxidase (exon nt numbers 76,163 – 76,310 to 77,520 – 77,821) has a 97% nt identity with the aldehyde oxidase of tomato (mRNA sequence, AF258814). Two PCR primers were designed (PALO2s/PALOs) to give an amplification of an intron, which is present in the S. demissum. PCR with this primer pair produced multiple bands, two bands of around 1.1 and 1.2 kb and one band of the expected size of 666 bp. The band of the expected size from G1h and S. habrochaites 1928 was cloned, despite repeated trials the band corresponding to HC7880 could not be cloned. This multiple band situation was not improved when annealing temperature was raised from 50 to 57oC and these primers were not used for further analyses.
TG30 Locus:
PCR primer pair (P30s/P30c) amplified a 500-bp fragment and the sequence was identical for HC7880, M82, Gh1, Gh2, Gh3, Gc9, Ih902, H24 (Ty2 gene), Gper11, CLN2443A (Ty2 gene), CLN2498-68. Thus, there is no evidence for an introgression from any wild species in this region of these lines.
TG26, T1660 and cLET24J2 region:
Primer design: The M13F TG26 sequence (complete sequence, App. I) had a 94%
and 96% nt identity for 189 nt with 46,465..46,654 and for 172 nt with 46,783..46,952,
respectively, of S. demissum BAC, AC136471 (123,428 nt). Two other RFLP probe
markers (cLET24J2 and T1660) also had high nt identity with this BAC clone sequence.
The sequence 10..450 of cLET24J2 had a 97% nt identity with 74,065..74,505 of
S. demissum and a 78% nt identity with the calmodulin mRNA (NM_101103). These
sequences also gave positive matches with A. thaliana (AC007190, gene At1g12310).
Two putative exons were identified in the S. demissum sequence; one corresponded
to 430..565 and 1..476 of cLET24J2 with 71,548..71,614 and 74,515..74,066, respectively,
for S. demissum. Two primers, PCAL1s and PACL1c, were designed to amplify the
putative intron with a fragment sequence of about 2,500 bp. For the T1660 marker
sequence, it had a 82% nt identity with A. thaliana (AF069299, 31,303..31,427),
which indicated the presence of an exon. Additionally, there was a high nt identity
of T1660 regions, 4..63, 66..141, and 143..279 with S. demissum AC136471 for
regions 70,226..70,286, 70070..70145, and 69030..69166, respectively. The A.
thaliana region corresponds to a putative ribosomal protein and the exons for
this gene were used to match regions of S. demissum (68,683..70,248) and then
design primers. This is the region of S. demissum BAC that corresponds to T1660
sequence. The two primers, PRib3s and PRib3c, were designed from the S. demissum
sequence (regions 69,110..69,132, and 69,130..70,110) that was conserved with
A. thaliana. Because the S. demissum BAC AC136471 was not annotated, the GlimmerM
(ver. 3) program at TIGR web site was used to detect putative ORFs. The ORFs
that appeared to be mostly likely genes as determined by length of exons over
1,000 bp were used to detect regions of high identity with A. thaliana. For
reference, the tomato RFLP marker probes sequences for TG26, T1660, and cLET24J2
correspond to nt number 46,600, 69,800, and 73,500, respectively. Putative ORF
#53 (77,727..79,701) had high identity with A. thaliana (AB018109, eg. 49,447..49,778)
and the exons from A. thaliana were compared with the sequence of S. demissum
and two primers designed (P53s and P53c), which would give a PCR fragment of
790 bp for S. demissum. Putative ORF #69 (117,424..119,162) had a high identity
(96%) with AF049900 of L. esculentum mRNA for gibberellin 20-oxidase and also
for A. thaliana (85%, AL161563). The mRNA sequence for L. esculentum was compared
with S. demissum and two sets of primers (P69s2/P69c2 and P69s1/P69c1) designed
from putative exons to amplify different introns.
Ribosomal Protein primers (Chromosome 11)
Fragments corresponding to HC7880, G1h and S. habrochaites 1928 were cloned and sequenced. The fragment corresponding to L. hirsutum 1928 was larger (around 900 bp) than the one from HC7880 and G1h (around 650 bp). These fragment sequences were aligned and it was found that 1928, which is “L. hirsutum” DNA, differs substantially from the L. esculentum sequence. There is for example a large intron which is missing in the L. esculentum sequence. There is one SNP between the Gh1 and HC7880, an A/G substitution. The HC7880 allele was designated G and the Gh1 allele g (Table 1).
Since two susceptible plants had the Gh1 allele, this region was not thought to be involved in resistance for the Gh1 phenotype.
Calmodulin protein primers (PCal1, Chromosome 11)
A PCR fragment of around 2.3 kb was amplified
from the parental lines, HC7880, Gh1, Gh2, Gh3 and 1928. An attempt was made
to clone this fragment, the digestion of plasmid DNA with EcoRI and BstZ1, however,
produced unexplainable results and this effort was abandoned.
TG26 locus:
PCR primers (P26s/P26c) fragments were directly sequenced. The following lines had the identical sequences: three resistant lines -Gh1, Gh2, Gh3, and one susceptible line M82. There were 16 SNPs between the sequence for these lines and S. habrochaites 1928, which does not support an introgression in this region from S. habrochaites in these lines. It would be very interesting to test H24 with this primer pair.
T1660 locus:
PCR primers (P69s2/P69c2) gave an 820 bp-fragment and this was directly sequenced. The sequence for the susceptible M82 and HC7880 lines was identical to the begomovirus resistant line G3h. Thus, there is no evidence for an introgression in this region. The abi sequence indicated that they are homozygous for this region. When the sequence was used in a BLAST search, a >97% nt identity was obtained for 8..174 and 745..820 with 784..950 and 949..1023 for the mRNA for gibberellin 20 oxidase-3 (AF04990). The intron region for tomato should be compared with the intron for S. demissum, which was used to design the primers.
P120 primers (Chromosome 11)
Three sets of primers corresponding to this region were designed, 120S-120ASC, 120S-120C and 120S-120AC. Only primer pair 120S-120ASC produced a single band (around 450 bp); this band, corresponding to parental genotypes HC7880, G1h, G2h and G3h, was sent for direct sequencing. Increasing the temperature of annealing to 570C with primer pair 120S-120AC resulted in the loss of the three bands that had been observed (0.4, 1.1 and 2 kb).
Probe 393 (Chromosome 11)
The PCR products corresponding to this probe (around 900 bp, primers P393s/P393c) were sequenced directly. No differences were observed between the parental lines HC7880, M82, G1h, G2h and G3h except G2h may have one indel at about 90 nt (an A).
Chromosome 6 with Ty1 gene:
For the Ty1 gene, clones of probes TG297 and TG436 in chromosome 6 that are located in the introgressed region from L. chilense in the resistant isoline TY52 (Theor. Appl. Genet. 88:141-146) (Fig. 3) were sequenced. TY52 and TY50, isoline to TY52 without the introgression, were supplied to Dani Zamir, Hebrew University of Jerusalem (HUJ). The RFLP map of the introgression was provided by H. Czosnek, which showed that introgression was TG97 and TG232.
For primer design, the RFLP probes CD67 (no sequence), TG97 (PstI, 1.4 kb, no good sequence), CT216c (EcoRI, 1.7 kb), TG232 (PstI, 1,415 bp), TG297 (PstI, 1,807 bp), TG436 (PstI, 1377 bp, HUJ; PstI, 1036 bp, Cornell University) were sequenced. Sequences for each probe were compared with data bases for Arabiopsis and GenBank. CT216c had one exon and one intron match with AT5g59550 (no primers designed). TG232 had 70% identity with for 28 – 496 nt with AC009991 (PTG232s1/PTG232c1). TG297 had an excellent match with AT5g22770 and 8 exons were identified (P297s3, P297s6, P297c6, P297c8). The sequence for the Cornell University probe TG436 matched exon 5 and 6 of At2g47680 (ATP-dependent RNA helicase A) (PCT436s5b/PCT436c6).
The most useful information was with PCR primer pairs for TG436 locus: PCT436s5b (5’ cct tcc aac ata cta tgc act tga gc 3’), PCT436c6 (5’ ccc aga aaa ctt gta aag aac ggc 3’). This primer pair gave a PCR fragment of about 900 bp, which was cloned and sequenced for S. habrochaites LA1223, G1h, TY52, TY50 and HC7880. Two indels were detected (Figure 4) among these five genotypes and TY52 and G1h shared the same indel pattern. As these were the two genotypes that had resistance to begomoviruses, two PCR primers were designed to amplify a 300-bp fragment (PSNP436s, 5’ tct gca agt cgc atc gga agg tat gc 3’; PSNP436c, 5’ gta tgg gcc acc tgg cat gca cct cg 3’) that would amplify the region with the two indels.
This primer pair was used to amplify PCR fragment, which was sequenced, from 10 additional genotypes. The genotypes were divided into two groups based on the two indels (Fig. 4). Those that have esculentum-type sequences and those that had the TY52 or chilense-type sequences. Those in the esculentum-type were TY50, HC7880, M82, G3h, GL11, CLN2443A (Ty 2 gene), CLN2498-68, and H24 (Ty 2 gene). The chilense-type sequence group included TY52 (Ty 1 gene), G1h, G2h, 902h, and G9c. G9c has resistance to TYLCV and ToMoV derived from S. chilense, so it may have the Ty 1 gene. Lines with resistance in Guatemala to begomoviruses were in both groups, which indicated that this region is not the location of the introgression from S. habrochaites, ie for G3h.
Because of the presence of the chilense-type sequence in two resistant lines from Guatemala, Gh1 and Gh2, the presence or absence of these two indels was determined in F3 populations from four crosses. Lines with resistance to begomoviruses Gh1 and Gh2 were crossed to susceptible parental lines HC-7880 and M82. The resulting F1 hybrids were planted at the beginning of 2002. The F2 populations were planted in early 2003 and resistant and susceptible plants (the most extreme phenotypes) were selected in March, 2003 (see web site for images: http://www.plantpath.wisc.edu/GeminivirusResistantTomatoes/CDR/Mar03/H16RS.htm). DNA was extracted from pooled tissue of the progeny of these plants (around 15 seedlings). This DNA, along with DNA from the parental lines, was used during this period (July, 2003) to screen for the indel marker in region 436 of Chromosome 6.
Two approaches were used to analyze the F3 populations. In one case, fluorescent-labeled primers were designed to be used with primers PSNP436s or PSNP436c for amplification of the sequence containing one of the two indels in that region. The PCR fragments were analyzed with a Gene Scan single lane system in order to determine their length; those with a deletion were expected to be shorter by two nucleotides. This is what was detected. The other approach involved the sequencing of PCR fragments from the primer pair PSNP436s/PSNP436c and scoring each sequence for esculentum-type, chilense-type or heterozygous. It was very easy to detect the heterozygous genotypes as the sequence would go out of frame exactly at the position of one indel and then into frame at the exact position of the second indel.
Results obtained with fluorescent (FAM) labeled primers and sequence data are
shown in Table 3. HC7880, M82 and Gh3 have genotype E/E (homozygous for esculentum
type, AA deletion followed by AC insertion). Gh1 and Gh2 have genotype e/e (homozygous,
chilense type, AA insertion followed by AC deletion). For the resistant phenotype,
there are 2, 13, and 5 plants in class E/E, E/e, and e/e, respectively. For
the susceptible plants, there are 7, 5, and 4 plants in class E/E, E/e, and
e/e, respectively. When the data are combined across both susceptible and resistant
phenotypes (9, 18, 9 plants for the E/e, E/e, e/e, respectively), the segregation
fits a 1:2:1 ratio, which would be expected for a random selection of plants.
Conclusion: Thus, the combined data are not supportive of a
linkage between the resistant phenotype in the Gh1 and Gh2 lines and the molecular
marker TG436. It is possible that these indels can be used as a marker for the
Ty-1 gene as they are present in TY52 and G9c.
RFLP – tagging of resistance genes to begomoviruses in a Guatemalan
breeding line
Luis Mejia, San Carlos University, M. J. Havey, J. Pritchard, and D. P. Maxwell, University of Wisconsin-Madison
Since no region of introgression had been detected for the resistance genes in Ih902 derived lines, an RFLP approach was also tried. DNA from two susceptible lines (HC7880 and M82) and one line (Gh1) resistant to the begomoviruses in Guatemala and derived from FAVI 9 (resistance genes from 902) were subjected to standard RFLP analysis with 33P-labelled probes for each of the 12 chromosomes. DNAs were digested with five restriction enzymes: DraI, EcoRI, EcoRV, HindIII, or XbaI. Chr1, top TG301, middle TG19; Chr2, top TG227; Chr3, middle TG129; Chr4, top TG146, middle TG62, bottom TG450 ; Chr5, bottom TG23, bottom CD74; Chr6, top TG231, TG119, middle TG54, bottom TG422; Chr7, top TG438, bottom CD54, Chr8, top TG41, middle TG282; bottom TG402; Chr9, top TG10, bottom TG8; Chr11, top TG194, bottom TG30, TG26 (TY2 gene introgression region); Chr12, middle CT79, bottom CD2.
Conclusions: No polymorphisms were detected for these 125 combinations of probes (25) and restriction enzymes (5).