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  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
  • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
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  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• This invention relates to the field of genetic manipulation of higher plants. More specifically, the invention relates to novel germplasms, breeding stocks and molecular markers created or identified by somatic fusion of domestic and wild potato species, which are useful for development of potato varieties resistant to late blight and other fungal pathogens.
• Classical breeding methods have been supplemented in recent years by molecular genetic techniques, e.g., to identify a gene that encodes a protein with antifungal or antibacterial properties (often not a plant gene) and then express this gene at high levels in a plant.
• Another approach is to use genes from wild species to improve disease resistance and other agronomic characteristics of cultivated crops. For the most part, however, breeders have been restricted to those genetic combinations that can be obtained by direct sexual crosses or through bridging crosses through several species.
• Protoplast fusion in some cases has provided a wider range of available genes.
• the somatic cells of two species are combined. Plants can then be regenerated from the combinations and examined for the expression of the desired attributes and for fertility.
• useful traits from widely separated, sexually incompatible, species into breeding lines.
• genes in those wild species responsible for conferring the useful trait such as resistance to one or more plant pathogens.
• the genes can then be incorporated into the genomes of a variety of different species to develop resistance to one or more plant pathogens.
• Potato Solanum tuberosum
• foliar diseases such as late blight and early blight
• virus diseases such as those caused by nematodes or Verticillium species
• bacterial diseases such as bacterial wilt (in the field) or Erwinia soft rot (in storage) .
• bacterial diseases such as bacterial wilt (in the field) or Erwinia soft rot (in storage) .
• These diseases are costly in terms of crop loss, expenses associated with application of chemicals and environmental impact of pesticide use. Such costs could be minimized or avoided if resistant potato varieties were available.
• adequate resistance for late blight, Erwinia soft rot and many other diseases has not been incorporated into potato cultivars, partly because of the lack of a good diversity of resistance genes that breeders can use to develop resistant cultivars.
• Late blight remains one of the most devastating diseases of potatoes worldwide. Despite its importance, no major cultivars with adequate late blight resistance are grown in the United states today. Until recently, crops were protected from the disease by cultural methods (e.g. crop rotation, crop hygiene) and with fungicides. The absence of compatible mating types within the U.S. heretofore has prevented sexual recombination. However, a second mating type has now become widespread in the U.S. and many lines of the fungus have become resistant to one of the key, very effective, systemic fungicides (Metalaxyl) registered for potato late blight control.
• Methodalaxyl very effective, systemic fungicides
• the late blight fungus is also a devastating pathogen on crops other than potato. It infects tomatoes, eggplants and other solanaceous species.
• Other Phytophthora species are pathogenic to a wide array of agronomically important plants, including grapes, avocados and several varieties of fruit and nut trees. Accordingly, a source of resistance to Phytophthora species that could be introduced into these species by molecular genetic techniques would also be of great value.
• Potato protoplasts have been fused with a number of sexually incompatible wild Solanum species (e.g., S . brevidens, S . bulbocastanum, S . commer ⁇ onii, S . polyadenium, S . etuburosum) , and many fertile somatic hybrids have been regenerated (Austin et. al., 1985, 1993; Ehlenfeldt & Helgeson, 1987; Kim-Lee et al., 1993; Novy & Helgeson, 1994b).
• S brevidens e.g., S brevidens, S . bulbocastanum, S . commer ⁇ onii, S . polyadenium, S . etuburosum
• Somatic hybrids have been screened for useful disease resistance (Helgeson et al., 1986; Austin et al., 1988; Novy & Helgeson, 1994b) and these resistances are heritable (Helgeson et al., 1993) .
• Solanum bulbocastanum is a particularly desirable wild species from which to seek useful disease resistance genetic traits, inasmuch as it exhibits resistance to several potato pathogens, including nematodes, early blight, late blight and Verticillium .
• Disease- or pest-resistant somatic hybrids of S . bulbocastanum and cultivated potato have been produced by the present inventors and by others.
• analysis of BC1 and BC2 progeny of a nematode-resistant S . bulbocastanum - S . tuberosum somatic hybrid revealed that the nematode resistance locus is likely to reside on chromosome 11 of S . bulbocastanum (Brown et al., 1996).
• the chromosomal location of late blight resistance in S . bulbocastanum heretofore has not been identified.
• This invention provides novel germplasms, breeding stocks, molecular markers and methods for introducing late blight resistance into cultivated potato plants.
• the invention further provides geno ic DNA segments from S . bulbocastanum useful for introducing resistance to the late blight fungus, Phytophthora infestans , into species other than potato.
• a potato germplasm confers resistance to the late blight fungus, Phytophthora infestans , as well as other fungal pathogens, including early blight, Erwinia soft rot and Verticillium .
• the most fundamental form of this germplasm is a tissue culture produced by somatic hybridization of S . tuberosum with S . bulbocastanum . Fertile plants regenerated from these hybrids are also provided, along with progeny resulting from crosses with agronomically preferable cultivated potato species.
• a late blight-resistant potato plant comprising a segment of a genome from Solanum bulbocastanum which contains a gene that confers resistance to late blight.
• the genomic segment of S . bulbocastanum is from chromosome 8, and co-segregates with one or more of the following markers: (1) a RAPD marker referred to herein as G02 586 ; (2) a RAPD marker referred to herein as P09 587 ; and (3) RFLP marker CT88, RFLP marker, RFLP marker CT148, RFLP marker CT252 and RFLP marker CT68.
• the potato plant may also be resistant to at least one additional disease, such as potato early blight, Erwinia soft rot, and Verticillium wilt.
• the aforementioned late blight resistance gene is incorporated into the potato plant by somatic hybridization between a cell of a parent of the plant and a cell of Solanum bulbocastanum .
• the late blight resistance gene is incorporated into the plant by genetic transformation of a cell of the plant with a plant transformation vector containing the gene.
• an isolated nucleic acid molecule is provided, which is complementary to part or all of a 0.6 kb segment of a Solanum bulbocastanum genome, which co-segregates with a gene that confers resistance to late blight.
• the segment comprises part or all of the RAPD marker G02 586 or P09 587 , having the sequence of SEQ ID NO:l or SEQ ID NO: 2, respectively.
• a method of monitoring late blight resistance in a breeding cross of progeny of a fertile somatic hybrid of Solanum tuberosum and Solanum bulbocastanum comprises: (a) performing the cross; (b) isolating genomic DNA from individual progeny of the cross; and (c) detecting in the genomic DNA the presence or absence of a genetic marker that is pre-determined to co-segregate with the late blight resistance, the presence or absence of the marker being indicative of the presence or absence of the late blight resistance in the individual progeny of the breeding cross.
• the marker is selected from the group of RAPD and RFLP markers listed above.
• a method of identifying a Solanum bulbocastanum gene that confers resistance to late blight comprises: (a) cloning a DNA segment that co-segregates with the late blight resistance phenotype in progeny of somatic hybrids of Solanum bulbocastanum and Solanum tuberosum ; (b) providing a genomic library of the Solanum bulbocastanum genome; (c) isolating clones of the genomic library that contain segments which hybridize with the co-segregating DNA segment; and (d) identifying at least one gene disposed within the isolated genomic clones that confers the late blight resistance.
• the cloned DNA segment that co-segregates with late blight resistance comprises part or all of one of the RAPD or RFLP markers listed above.
• a late blight resistance gene from Solanum bulbocastanum produced by the aforementioned method, is provided. Also provided is a transgenic plant comprising the resistance gene.
• FIG. 1 RFLP analysis of somatic hybrids between S . bulbocastanum and potato.
• the probe used in the analysis was TG310, a tomato genomic probe specific for Chromosome 1 of tomato and potato.
• Figure 2. MapMaker analysis of chromosome 8 of
• Solanum bulbocastanum with RAPD randomly amplified polymorphic DNA
• RFLP restriction fragment length polymorphism
• Percentages in parentheses (left column) indicate recombination frequencies calculated by dividing the deviation from complete co-inheritance by the population size. Numbers to the right of the percentage column indicate distances between markers in centimorgans. Numbers in parentheses to the right of the chromosome 8 diagram represent the arbitrary code number of the individual in the population.
• the far right column lists RAPD markers, which are named by the decameric primer and the size of the amplified transcript (e.g. "G02-586" or "P09-587") and RFLP markers.
• the resistance locus is indicated by "R” . Map scale is 10.0 cM per 1.21 cm.
• BC 1 progeny were crossed with three different potato breeding lines (Norland, Atlantic and A 89804-7) , all of which are sensitive to the late blight fungus.
• Each BC 2 population contained more than 50 individuals and segregated for resistance to late blight.
• late blight resistance was correlated (>95%) with the presence of a RAPD marker ("G02 586 ”) keyed to chromosome 8 of S . bulbocastanum .
• G02 586 RAPD marker
• This invention provides a new and useful germplasm and breeding stock for breeding potato cultivars with resistance to late blight.
• the germplasm comprises fertile hybrids produced by somatic fusion of S . bulbocastanum with S . tuberosum , and progeny thereof, which contain that portion of the S . bulbocastanum genome carrying the late blight resistance gene or genes.
• the presence of this genomic fragment is conveniently monitored by the presence of closely linked RAPD markers, G02 586 or P09 587 , or closely-linked RFLP markers, such as CT88, as described in greater detail below.
• Particularly preferred breeding stock is obtained by repeated backcrosses of the somatic hybrid with potato cultivars having desirable agronomic qualities, with the presence of the late blight resistance-conferring genomic segment of S . bulbocastanum being monitored by detection of one or more of the relevant RAPD or RFLP markers.
• Creation of S . tuberosum - S . bulbocastanum somatic hybrids, selection of fertile, resistant plants, and production of subsequent backcross generations comprising the disease resistance gene or genes are all accomplished by methods well known to plant breeders and molecular biologists. Preferred methods are described in greater detail in Example 1.
• This invention also provides novel molecular markers to facilitate selection of breeding progeny that contain the resistance-conferring genomic segment from S . bulbocastanum, without having to perform field or greenhouse trials for disease resistance.
• One closely linked marker, G02 586 was created through the use of RAPD markers, using commercially available oligonucleotide 10- mers as primers for PCR amplification.
• RAPD markers are generated by incubating genomic DNA with a population of 10-mers under conditions that allow the oligonucleotides to bind to any complementary sequences in the genomic DNA.
• the length of DNA between two bound sets of primers is amplified by PCR, thereby generating a DNA segment which is a copy of the segment between the primers in the genomic DNA, i.e. a RAPD fragment.
• the nucleotide sequence of the G02 586 RAPD marker is set forth herein as SEQ ID N0:1.
• RAPD fragments thus created are species specific markers which can be keyed to particular chromosomes by comparative RFLP analysis and can be followed as dominant markers through various crosses.
• RAPD marker G02 586 (which is a 586 bp fragment primed by the "G02" decameric oligonucleotide purchased from Operon Technologies, Inc.) was keyed to S . bulbocastanum chromosome 8.
• Analysis of BC 2 progeny of potato-S. bulbocastanum somatic hybrids demonstrated that late blight resistance correlates with greater than 95% frequency with the presence of the G02 586 RAPD fragment.
• This invention also provides a second RAPD marker, P09 587 , which is also closely linked with the resistance gene(s) in S . bulbocastanum , and which was identified by the same protocol as set forth above for G02 586 .
• the nucleotide sequence of RAPD marker P09 587 is set forth herein as SEQ ID NO: 2.
• This invention further provides RFLP molecular markers, useful in facilitating selection of breeding progeny that contain the resistance-conferring segment of S . bulbocastanum . These markers can also assist in defining the location of the resistance genes on the chromosome, and obtaining isolated genomic segments containing the gene(s).
• the nucleotide sequence of RFLP CT88 from three different sources (published by Tanksley et al. (http: //probe. nalusda.gov: 8300/cgi- bin/browse/solgenes) , from R4 potato, and from S . bulbocastanum) are set forth herein as SEQ ID NOS: 3, 4 and 5 respectively) .
• useful RAPD or RFLP fragments can be maintained in any suitable cloning vector.
• the G02 586 marker generated in accordance with the present invention is maintained in a plasmid vector provided with a commercially available PCR kit (Invitrogen, Inc.). It is noted that obtaining RAPD should be replicable by anyone of skill in the art, using the commercially available decamers and the methods described in Example 2 and references cited therein.
• RAPD fragments described herein can be duplicated by nucleotide synthesis using standard methodologies.
• the RAPD fragments, or portions thereof, or any of the linked RFLPs discussed above or shown in Figure 2 are used to monitor the presence or absence of the late blight resistance gene(s) by labeling them as probes to hybridize with complementary sequences in the S . bulbocastanum genome.
• the cloned fragments may be labeled according to any standard means, many of which are set forth in "Current Protocols in Molecular Biology", ed ⁇ . Frederick M. Ausubel et al., John Wiley & Sons, 1997.
• the complementary genomic DNA is detected by one of several standard methods including, but not limited to, (1) in situ hybridization; (2) Southern hybridization (3) "dot blot” hybridization; and (4) assorted amplification reactions such as polymerase chain reactions (PCR) . Detection of the complementary genomic DNA is indicative of the presence of the late blight resistance-conferring gene(s) , due to the close linkage of the R gene(s) and the RAPD marker or RFLPs, as discussed above.
• the RAPD fragments, or portions thereof, or any of the aforementioned RFLP fragments may also be used to identify and isolate the closely-linked late blight resistance gene of S . bulbocastanum , using methods well known to molecular biologists.
• a genomic library of S . bulbocastanum is constructed a suitable cloning vehicle, e.g. cosmid, yeast artificial chromosome (YAC) or bacterial artificial chromosome (BAC) .
• YAC yeast artificial chromosome
• BAC bacterial artificial chromosome
• the library is then screened by hybridization with the cloned RAPD fragment and/or one or more of the RFLP markers, and hybridizing clones are isolated.
• the hybridizing clones are then further analyzed, e.g.
• candidate open reading frames that may encode the resistance factor (s).
• candidate open reading frames Once candidate open reading frames are identified, they in turn may be further analyzed (e.g. by construction and in vitro expression of a cDNA molecule) in order to characterize the resistance gene and its encoded protein.
• the disease resistance-conferring clone thus identified may be used to introduce late blight resistance into cultivated potato by molecular genetic techniques.
• a binary bacterial artificial chromosome (BIBAC) vector may be used to mobilize a BAC genomic insert into potato (the vector BIBAC2 (Hamilton et al, 1996) has been used to mobilize large (>150 kb) DNA inserts into tobacco) .
• a BIBAC vector containing the relevant genomic insert from S . bulbocastanum can be transferred into an Agrobacterium tumefaciens and used to transform selected potato cultivars.
• potato cells can be transformed via biolistic delivery of the BIBAC clone. Putative transgenic clones can then be evaluated by standard methods to determine if the transformation has been successful.
• the disease resistance-conferring clone also may be used to introduce resistance to Phytophthora infestans in species other than potato, which are sensitive to infection by that organism. These species include, but are not limited to, tomato, eggplant and other Solanum species.
• the late blight resistance gene(s) of S . bulbocastanum may confer resistance to diseases other than late blight, and so may be of even broader utility for introducing disease resistance in potato and other plant species.
• the gene may be particularly useful for introducing resistance to other Phytophthora species pathogenic to grapes, avocados, fruit trees and nut trees. Moreover, once the gene's function has been determined, this may lead to the discernment of new mechanisms of resistance in other species.
• Solanum bulbocastanum and Potato, and Progeny Thereof A Mexican wild species, S . bulbocastanum, is highly resistant to late blight. However, S . bulbocastanum is a 1EBN species and thus extremely difficult to cross directly with potato.
• Somatic hybridization can provide a means of bypassing sexual incompatibility between Solanum species, leading to fertile plants that can be used directly in breeding programs.
• the experimental results set forth in this Example demonstrate that the resistance in S . bulbocastanum can be captured and passed on to potato breeding lines by the use of somatic hybridization.
• Potato and related species used for somatic hybridization were obtained from Dr. John Bamberg and his colleagues at the Inter-Regional Potato Introduction Station (NRSP-6) , 4312 Highway 42, Sturgeon Bay, WI . These include S . bulbocastanum , PI 243510 and S . tuberosum PI 23900 (potato) .
• NRSP-6 Inter-Regional Potato Introduction Station
• S . bulbocastanum 4312 Highway 42, Sturgeon Bay, WI
• S PI 243510 include S 243510 and S . tuberosum PI 23900 (potato) .
• Elite copies of potato cultivars were obtained from the Wisconsin potato certification program. All cultivars and wild species, and test materials were routinely maintained clonally in vitro as described by Haberlach et al (1985). The individual clones were multiplied in vitro for analyses.
• Protoplasts were isolated from leaves of in vitro shoots as described by Haberlach et al. (1985). Somatic hybridizations with the protoplasts were performed using a polyethylene glycol (PEG) protocol. For the most part, the procedure of Austin et al. (1985) was followed. However, after PEG additions, dilutions and pelleting of the cells after the fusion attempts, the cells were suspended in 0.3 M sucrose rather than 0.6 M mannitol. The cell suspension was gently shaken (40 RPM) for 45 minutes and then centrifuged (HNII centrifuge, IEC) at 1300 rpm for 10 minutes in a Babcock bottle. This modification resulted in viable protoplasts and fused cells being concentrated at the surface of the sucrose solution in the bottle, thus separating the viable cells from the pelleted debris.
• PEG polyethylene glycol
• the resulting fused cells were regenerated into whole plants in a manner similar to that reported by Haberlach et al. (1985) .
• the cells were plated onto culture medium (CUL, Haberlach et al. 1985) and, after macroscopic calli had appeared, the calli were transferred to differentiation medium ( DIF, Haberlach et al. 1985). After 2-3 weeks, the calli were transferred to the differentiation medium developed by Lam (1977) . After buds had formed, the calli were transferred to proliferation medium (PM, Haberlach et al. 1985) .
• Shoots that formed were then excised and rooted on standard propagation medium (PROP, Haberlach et al. 1985) and maintained in test tubes in vitro . Clonal copies of the reference copy were made for experiments.
• RFLP restriction fragment length polymorphism
• J101, J103, and J138 Three of the hybrids have been used extensively in further experiments. These were designated J101, J103, and J138.
• the potato parent was designated with K (for Katahdin) or A (for Atlantic) .
• J101K1 was the first seed germinated from a berry obtained from the cross of J101 and Katahdin.
• J101K6 and J101K27 were the seedlings obtained from seed 6 and seed 27 respectively from that cross.
• the BC2 progeny were named by adding the seed number and cultivar lines in that cross.
• the cross designated as J101K6A22 is the 22nd seedling from the cross of line J101K6 with Atlantic. This shortcut avoided use of the longer and less informational term ( (S . bulbocastanum + S .
• BC 1 and BC 2 lines were tested in Toluca, Mexico in a second growing season the following summer. Good resistance in the Toluca field test was also obtained with all lines that were resistant in Wisconsin in the previous summer.
• the resistance to P . infestans from S . bulbocastanum appears to be more general than the race- specific resistance derived from S . demis ⁇ um (Black & Gallegly, 1957) . Nearly every race of the fungus is known to be found in Toluca, Mexico and was actually isolated from the fields where the progeny of the somatic hybrids showed good resistance. Observations of the foliage in Toluca in two different growing seasons indicated that some lesions actually formed and that limited sporulation also occurred. Although the disease resistance is highly effective, it is unclear, as yet, the numbers of genes involved. For each of the somatic hybrids tested, the disease resistances of BC, lines appear to segregate. Thus, it appears that the late blight resistance gene(s) is (are) heterozygous in the clone of S . bulbocastanum utilized in somatic hybridization.
• Example 1 described the production of potato-S. jbuljbocastanujn somatic hybrids and their progeny, which are resistant to potato late blight. This example describes the identification of DNA markers that co- segregate with disease resistance in these progeny.
• RFLP restriction fragment length polymorphism
• the procedure usually generates between about five and eight specific bands that can be followed, as dominant markers, through various crosses. Moreover, if specific bands can be linked to a characteristic (such as disease resistance) or to a particular chromosome, or both, the bands can be excised and amplified, then used as a standard RFLP.
• the method works well to evaluate the extension of DNA introgression from a wild species into a cultivated species.
• RAPDs are species specific, so it is necessary to develop a set for each different species, unlike with RFLPs where inter-species synteny applies.
• Somatic hybrids S . bulbocastanum (PI 243510) and S . tuberosum (PI 203900) were generated by the method of Austin et al. (1985 and 1993), as describe in detail in Example 1. Plants were derived from fertile hybrids, and three lines of these were designated J101, J103 and J138, respectively. The somatic hybrid plants were crossed as the female parent with S . tuberosum cv Katahdin (KAT) to give BC 1 progeny. Three BC 1 progeny were crossed as seed parents with three different potato breeding lines (Norland, Atlantic and A 89804-7) to generate BC 2 populations.
• RAPD markers were named by the deca- nucleotide primer (obtained from Operon Technologies) and the size of the amplified fragment in subscript (e.g., a 586 bp fragment amplified by primer G02 is represented as G02 586 ) . Segregation of RAPD markers was also analyzed with maximum likelihood algorithms contained in the MapMaker computer package (Lander et al., 1987). MapMaker version 2.0 for the Macintosh was used. The data were coded and analyzed as a "Haploid" population under the "Data Type” option. The use of MapMaker for the analysis of interspecific somatic hybrids is non- standard and does not provide three-point linkage data.
• recombination frequencies do have meaning in this context. Markers that show identical segregation have a recombination frequency of 0.0%. Markers that deviate from complete co-inheritance by a single change (e.g. a marker is present in one additional individual) show a recombination frequency proportional to the population size; in this instance 1/101 individuals or 1.0%. Thus, multiples of this value indicate the number of differences observed between any pair of markers.
• SEQ ID NO: 1 The nucleotide sequence of G02 586 is set forth herein as SEQ ID NO: 1.
• the nucleotide sequence of P09 587 is set forth herein as SEQ ID NO: 2.
• Three nucleotide sequences of CT88 are set forth herein.
• SEQ ID NO: 3 is the sequence published by Tanksley et al. (http: //probe. nalusda.gov.8300/cgi-bin/browse/solgenes) ;
• SEQ ID NO: 4 is from R4 potato, and
• SEQ ID NO: 5 is from S. bulbocastanum (PT29) . Slight differences were noted among the three sequences.
• the R4 potato marker is 589 bp in length, while the S .
• bulbocastanum RFLP is 592 bp and the Tanksley et al. sequence is 596 bp.
• S . bulbocastanum CT88 homolog possesses two TaqI sites, whereas the other two have only one.
• the fusion of S . tuberosum with S . bulbocastanum yielded 17 confirmed somatic hybrids.
• the somatic hybrids are quite resistant to early blight and to late blight.
• the progeny of some crosses were found to segregate for high resistance to both early blight and late blight. Some of the progeny are highly resistant even to fungal lines that are highly virulent, complex races. Several highly resistant clones were selected and further crossed with potato.
• Example 1 and BC2 populations Segregation of late blight resistance in selected BC 1 and BC2 populations were shown in Example 1, Tables 1 and 2.
• Table 3 shows results of segregation analysis of BC 2 progeny with respect to late blight resistance in relation to the presence or absence of RAPD marker G02 586 for Chromosome 8 of S . bulbocastanum .
• the data shown in Table 3 were generated by PCR amplification, followed by agarose gel electrophoresis and ethidium bromide staining to observe the presence or absence of an amplified 586 bp band.
• Table 3 Segregation of BC2 progeny in relation to the presence or absence of RAPD marker G02 586 .
• late blight resistance in the BC 2 clones is highly correlated (>95%) with the presence of RAPD marker G02 586 , which has been keyed to chromosome 8 of S . bulbocastanum .
• This high correlation of the resistance phenotype with the marker indicates that the gene(s) responsible for conferring late blight resistance reside on the chromosome at or near the G02 586 marker.
• the marker can be used as a molecular tag to follow resistance through breeding programs and to identify and isolate disease-resistant breeding stock. Additionally, knowing the relative proximity of the resistance gene(s) to the marker, isolation of the gene(s) will be facilitated.
• the RFLP and RAPD markers CT88 and P09 587 discussed above can also be used as molecular tags to follow resistance through breeding programs.
• the use of a combination of the resistance-associated RAPD and RFLP markers can provide an added advantage in following segregation in breeding, and eventually in isolating and cloning the S . bulbocastanum gene(s) responsible for conferring resistance to late blight and other diseases.
• Fertile interspecific somatic hybrids of Solanum A novel source of resistance to Erwinia soft rot.
• MAPMAKER an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1: 174-181.

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  • 1998-07-27 CN CN98809716A patent/CN1314782A/zh active Pending
  • 1998-07-27 AU AU86762/98A patent/AU8676298A/en not_active Abandoned
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  • 1998-07-27 EP EP98938178A patent/EP1003364A1/en not_active Withdrawn
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