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Identification of a resistance gene Rpi-dlc1 to Phytophthora infestans in European accessions of Solanum dulcamara.

Golas TM, Sikkema A, Gros J, Feron RM, van den Berg RG, van der Weerden GM, Mariani C, Allefs JJ - Theor. Appl. Genet. (2009)

Bottom Line: Blast analysis of the sequenced markers resulted in a plausible gene position on the distal end of the long arm of chromosome 9 that could be confirmed by CAPS markers.In addition, one population was tested for broadness of resistance responses using a set of seven additional P. infestans isolates, varying in virulence.This indicated the possible presence of additional Rpi genes.

View Article: PubMed Central - PubMed

Affiliation: Plant Cell Biology Group, Radboud University, Nijmegen, The Netherlands. T.golas@science.ru.nl

ABSTRACT
Initial screening of 14 Solanum dulcamara accessions enabled the identification of individuals resistant and susceptible to Phytophthora infestans. Crosses between contrasting genotypes resulted in three F(2)-BC(1) populations segregating for resistance to late blight in a laboratory assay and under field conditions. Genetic profiling of one of these populations using 128 AFLP primers generated three markers linked to the resistant phenotype. Blast analysis of the sequenced markers resulted in a plausible gene position on the distal end of the long arm of chromosome 9 that could be confirmed by CAPS markers. Thus, we describe a first resistant gene, named Rpi-dlc1, from S. dulcamara, a Solanum species native to Europe. In addition, one population was tested for broadness of resistance responses using a set of seven additional P. infestans isolates, varying in virulence. This indicated the possible presence of additional Rpi genes.

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a Maps of chromosome 9 of potato (RH × SH) and S. dulcamara near the Rpi-dlc1 locus. Numbers indicate genetic distances in cM between markers. b Schematic representation of the genomic region in the population 05-346 containing Rpi-dlc1.Black bars represent resistant, white bars susceptible marker profiles and gray bars indicate the genomic region where Rpi-dlc1 was mapped. At the bottom end of the bars results from the field experiment are presented. Numbers indicate number of offspring with clear phenotype, R is resistant, S susceptible and n.d. not determined phenotype. Rp and Sp are the resistant and susceptible parents, respectively. Genetic distances between markers GP101 and T0521 were calculated for 302 individuals. Among these, 23 recombinants were found. Genetic distances for the remaining three polymorphic markers S1d5-a, GP41 and S1d11 were calculated using 210 individuals and 16 recombinants that were tested in the field. On the right a cM scale
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Fig2: a Maps of chromosome 9 of potato (RH × SH) and S. dulcamara near the Rpi-dlc1 locus. Numbers indicate genetic distances in cM between markers. b Schematic representation of the genomic region in the population 05-346 containing Rpi-dlc1.Black bars represent resistant, white bars susceptible marker profiles and gray bars indicate the genomic region where Rpi-dlc1 was mapped. At the bottom end of the bars results from the field experiment are presented. Numbers indicate number of offspring with clear phenotype, R is resistant, S susceptible and n.d. not determined phenotype. Rp and Sp are the resistant and susceptible parents, respectively. Genetic distances between markers GP101 and T0521 were calculated for 302 individuals. Among these, 23 recombinants were found. Genetic distances for the remaining three polymorphic markers S1d5-a, GP41 and S1d11 were calculated using 210 individuals and 16 recombinants that were tested in the field. On the right a cM scale

Mentions: Amplified fragment length polymorphism analyses were conducted on population 05-203 and this ultimately yielded three co-segregating fragments: two at 5 cM, and one fragment at 2 cM distance from a gene that putatively was designated Rpi-dlc1. Primers were designed based on the sequence of these AFLP markers and tested on a pre-amplification mix to confirm whether the proper fragments were isolated. Two out of three primers pairs reproduced the original segregation pattern of the AFLP markers, thereby confirming correct cloning. These two confirmed markers (eACTmCAC with primer pair 5′–3′-catggcttcccgtactgaat and 5′–3′-tcacctttccaggcaaaaac and eACTmCGC 5′–3′-cgcacaatttgtgcatcgcg and 5′–3′-cactgtagtagcatatttgg) were tested on genomic DNA of population 05-203 and both amplified a product of the expected size in all individuals, but the amount of PCR product was considerably lower in the susceptible parent, susceptible offspring and recombinants. As a next step toward the mapping of Rpi-dlc1, the sequences of the markers eACTmCAC and eACTmCGC were blasted against plant genomic sequences available in public databases. The highest sequence homology of the AFLP fragment amplified by eACTmCAC (accession number: FJ769334) was with the S-adenosyl-l-homocysteine hydrolase gene from S. tuberosum, found in the GABI database, indicating two possible positions for Rpi-dlc1: one on potato chromosome 9 between markers GP41 and CT220 and the other on chromosome 12 between markers GP122 and GP264. To verify both putative genomic positions of Rpi-dlc1, four primer pairs were developed based on the available sequences of these markers. Pairs yielding clear PCR products were used to search for polymorphisms between the parental genotypes of population 05-203. Therefore, PCR products were digested with a set of 12 restriction enzymes. As a first result, marker GP41, after digestion with RsaI, revealed a polymorphism between the parents of population 05-203. Subsequently, GP41/RsaI was tested on the entire population 05-203 and was indeed found to co-segregate with the phenotypic scores. Positions on chromosome 12 did not yield a polymorphic band. Subsequently from available databases, a set of 28 markers, previously mapped in the region of GP41 in potato or tomato, were selected and primers were developed on the basis of their available sequences. In total, 16 primer pairs yielded amplification products in S. dulcamara and 4 of them (S2g3/AluI; TG591A-L/BsuRI; CT220est/NlaIII; S1d11/allele specific) were polymorphic and co-segregated with resistance in population 05-203 (Table 3). The other two segregating populations, 05-188 and 05-346, were also tested for co-segregation at the Rpi-dlc1 locus with the set of markers used in population 05-203 (Table 3). In both populations, segregation of the identified polymorphisms was consistent with phenotypic data. In population 05-346, originally consisting of 52 offspring clones, recombinants were identified between Rpi-dlc1 and a set of five available polymorphic markers. This population was subsequently enlarged to 302 individuals to generate a more detailed map of the Rpi-dlc1 locus. In total, 23 crossing-over events were identified between markers GP101 and the T0521 marker (7.6 cM). T0521 is positioned more distal to the centromere than marker GP101. However, due to the loss of 7 of these recombinant genotypes, only 16 could eventually be phenotyped in the field and tested with the available remaining three polymorphic markers. To be able to compare the genetic distances obtained for GP101 and T0521, genetic distances between S1d5-a, GP41 and S1d11 were calculated for 210 individuals/16 recombinants (Fig. 2b). In the enlarged population 05-346 each marker appeared to be separated by at least one crossing over. This allowed narrowing down the genetic region containing Rpi-dlc1 by comparing the presence/absence of a marker with resistance/susceptibility of the recombinant. Between markers S1d11 and T0521, one resistant recombinant was identified containing all markers except T0521, excluding the possible presence of Rpi-dlc1 near T0521. Recombinant containing T0521 and S1d11 could not be used due to the lack of reliable phenotypic results. Subsequently, three resistant recombinants were identified that contained GP101, S1d5-a and GP41 markers, but not S1d11. In addition, a susceptible recombinant was identified that contained GP41, S1d11 and T0521, but not GP101 and S1d5-a, thus excluding the GP41 region. Finally two sets of recombinants; one containing only GP101 and second containing all other markers except GP101 were found to contain both resistant and susceptible genotypes, indicating that the Rpi-dlc1 locus is localized somewhere between the GP101 and S1d5-1 markers (Fig. 2b).Table 3


Identification of a resistance gene Rpi-dlc1 to Phytophthora infestans in European accessions of Solanum dulcamara.

Golas TM, Sikkema A, Gros J, Feron RM, van den Berg RG, van der Weerden GM, Mariani C, Allefs JJ - Theor. Appl. Genet. (2009)

a Maps of chromosome 9 of potato (RH × SH) and S. dulcamara near the Rpi-dlc1 locus. Numbers indicate genetic distances in cM between markers. b Schematic representation of the genomic region in the population 05-346 containing Rpi-dlc1.Black bars represent resistant, white bars susceptible marker profiles and gray bars indicate the genomic region where Rpi-dlc1 was mapped. At the bottom end of the bars results from the field experiment are presented. Numbers indicate number of offspring with clear phenotype, R is resistant, S susceptible and n.d. not determined phenotype. Rp and Sp are the resistant and susceptible parents, respectively. Genetic distances between markers GP101 and T0521 were calculated for 302 individuals. Among these, 23 recombinants were found. Genetic distances for the remaining three polymorphic markers S1d5-a, GP41 and S1d11 were calculated using 210 individuals and 16 recombinants that were tested in the field. On the right a cM scale
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Related In: Results  -  Collection

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Fig2: a Maps of chromosome 9 of potato (RH × SH) and S. dulcamara near the Rpi-dlc1 locus. Numbers indicate genetic distances in cM between markers. b Schematic representation of the genomic region in the population 05-346 containing Rpi-dlc1.Black bars represent resistant, white bars susceptible marker profiles and gray bars indicate the genomic region where Rpi-dlc1 was mapped. At the bottom end of the bars results from the field experiment are presented. Numbers indicate number of offspring with clear phenotype, R is resistant, S susceptible and n.d. not determined phenotype. Rp and Sp are the resistant and susceptible parents, respectively. Genetic distances between markers GP101 and T0521 were calculated for 302 individuals. Among these, 23 recombinants were found. Genetic distances for the remaining three polymorphic markers S1d5-a, GP41 and S1d11 were calculated using 210 individuals and 16 recombinants that were tested in the field. On the right a cM scale
Mentions: Amplified fragment length polymorphism analyses were conducted on population 05-203 and this ultimately yielded three co-segregating fragments: two at 5 cM, and one fragment at 2 cM distance from a gene that putatively was designated Rpi-dlc1. Primers were designed based on the sequence of these AFLP markers and tested on a pre-amplification mix to confirm whether the proper fragments were isolated. Two out of three primers pairs reproduced the original segregation pattern of the AFLP markers, thereby confirming correct cloning. These two confirmed markers (eACTmCAC with primer pair 5′–3′-catggcttcccgtactgaat and 5′–3′-tcacctttccaggcaaaaac and eACTmCGC 5′–3′-cgcacaatttgtgcatcgcg and 5′–3′-cactgtagtagcatatttgg) were tested on genomic DNA of population 05-203 and both amplified a product of the expected size in all individuals, but the amount of PCR product was considerably lower in the susceptible parent, susceptible offspring and recombinants. As a next step toward the mapping of Rpi-dlc1, the sequences of the markers eACTmCAC and eACTmCGC were blasted against plant genomic sequences available in public databases. The highest sequence homology of the AFLP fragment amplified by eACTmCAC (accession number: FJ769334) was with the S-adenosyl-l-homocysteine hydrolase gene from S. tuberosum, found in the GABI database, indicating two possible positions for Rpi-dlc1: one on potato chromosome 9 between markers GP41 and CT220 and the other on chromosome 12 between markers GP122 and GP264. To verify both putative genomic positions of Rpi-dlc1, four primer pairs were developed based on the available sequences of these markers. Pairs yielding clear PCR products were used to search for polymorphisms between the parental genotypes of population 05-203. Therefore, PCR products were digested with a set of 12 restriction enzymes. As a first result, marker GP41, after digestion with RsaI, revealed a polymorphism between the parents of population 05-203. Subsequently, GP41/RsaI was tested on the entire population 05-203 and was indeed found to co-segregate with the phenotypic scores. Positions on chromosome 12 did not yield a polymorphic band. Subsequently from available databases, a set of 28 markers, previously mapped in the region of GP41 in potato or tomato, were selected and primers were developed on the basis of their available sequences. In total, 16 primer pairs yielded amplification products in S. dulcamara and 4 of them (S2g3/AluI; TG591A-L/BsuRI; CT220est/NlaIII; S1d11/allele specific) were polymorphic and co-segregated with resistance in population 05-203 (Table 3). The other two segregating populations, 05-188 and 05-346, were also tested for co-segregation at the Rpi-dlc1 locus with the set of markers used in population 05-203 (Table 3). In both populations, segregation of the identified polymorphisms was consistent with phenotypic data. In population 05-346, originally consisting of 52 offspring clones, recombinants were identified between Rpi-dlc1 and a set of five available polymorphic markers. This population was subsequently enlarged to 302 individuals to generate a more detailed map of the Rpi-dlc1 locus. In total, 23 crossing-over events were identified between markers GP101 and the T0521 marker (7.6 cM). T0521 is positioned more distal to the centromere than marker GP101. However, due to the loss of 7 of these recombinant genotypes, only 16 could eventually be phenotyped in the field and tested with the available remaining three polymorphic markers. To be able to compare the genetic distances obtained for GP101 and T0521, genetic distances between S1d5-a, GP41 and S1d11 were calculated for 210 individuals/16 recombinants (Fig. 2b). In the enlarged population 05-346 each marker appeared to be separated by at least one crossing over. This allowed narrowing down the genetic region containing Rpi-dlc1 by comparing the presence/absence of a marker with resistance/susceptibility of the recombinant. Between markers S1d11 and T0521, one resistant recombinant was identified containing all markers except T0521, excluding the possible presence of Rpi-dlc1 near T0521. Recombinant containing T0521 and S1d11 could not be used due to the lack of reliable phenotypic results. Subsequently, three resistant recombinants were identified that contained GP101, S1d5-a and GP41 markers, but not S1d11. In addition, a susceptible recombinant was identified that contained GP41, S1d11 and T0521, but not GP101 and S1d5-a, thus excluding the GP41 region. Finally two sets of recombinants; one containing only GP101 and second containing all other markers except GP101 were found to contain both resistant and susceptible genotypes, indicating that the Rpi-dlc1 locus is localized somewhere between the GP101 and S1d5-1 markers (Fig. 2b).Table 3

Bottom Line: Blast analysis of the sequenced markers resulted in a plausible gene position on the distal end of the long arm of chromosome 9 that could be confirmed by CAPS markers.In addition, one population was tested for broadness of resistance responses using a set of seven additional P. infestans isolates, varying in virulence.This indicated the possible presence of additional Rpi genes.

View Article: PubMed Central - PubMed

Affiliation: Plant Cell Biology Group, Radboud University, Nijmegen, The Netherlands. T.golas@science.ru.nl

ABSTRACT
Initial screening of 14 Solanum dulcamara accessions enabled the identification of individuals resistant and susceptible to Phytophthora infestans. Crosses between contrasting genotypes resulted in three F(2)-BC(1) populations segregating for resistance to late blight in a laboratory assay and under field conditions. Genetic profiling of one of these populations using 128 AFLP primers generated three markers linked to the resistant phenotype. Blast analysis of the sequenced markers resulted in a plausible gene position on the distal end of the long arm of chromosome 9 that could be confirmed by CAPS markers. Thus, we describe a first resistant gene, named Rpi-dlc1, from S. dulcamara, a Solanum species native to Europe. In addition, one population was tested for broadness of resistance responses using a set of seven additional P. infestans isolates, varying in virulence. This indicated the possible presence of additional Rpi genes.

Show MeSH
Related in: MedlinePlus