Isolation of molecular markers for tomato (L. esculentum) using random amplified polymorphic DNA (RAPD)

Summary A new DNA polymorphism assay was developed in 1990 that is based on the amplification by the polymerase chain reaction (PCR) of random DNA segments, using single primers of arbitrary nucleotide sequence. The amplified DNA fragments, referred to as RAPD markers, were shown to be highly useful in the construction of genetic maps (RAPD mapping). We have now adapted the RAPD assay to tomato. Using a set of 11 oligonucleotide decamer primers, each primer directed the amplification of a genome-specific fingerprint of DNA fragments. The potential of the original RAPD assay to generate polymorphic DNA markers with a given set of primers was further increased by combining two primers in a single PCR. By comparing fingerprints of L. esculentum, L. pennellii, and the L. esculentum chromosome 6 substitution line LA1641, which carries chromosome 6 from L. pennellii, three chromosome 6-specific RAPD markers could be directly identified among the set of amplified DNA fragments. Their chromosomal position on the classical genetic map of tomato was subsequently established by restriction fragment length polymorphism (RFLP) linkage analysis. One of the RAPD markers was found to be tightly linked to the nematode resistance gene Mi.     

Comparison of RFLP and RAPD markers to estimating genetic relationships within and among cruciferous species

Restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers are being used widely for evaluating genetic relationships of crop germplasm. Differences in the properties of these two markers could result in different estimates of genetic relationships among some accessions. Nuclear RFLP markers detected by genomic DNA and cDNA clones and RAPD markers were compared for evaluating genetic relationships among 18 accessions from six cultivated Brassica species and one accession from Raphanus sativus. Based on comparisons of genetic-similarity matrices and cophenetic values, RAPD markers were very similar to RFLP markers for estimating intraspecific genetic relationships; however, the two marker types gave different results for interspecific genetic relationships. The presence of amplified mitochondrial and chloroplast DNA fragments in the RAPD data set did not appear to account for differences in RAPD- and RFLP-based dendrograms. However, hybridization tests of RAPD fragments with similar molecular weights demonstrated that some fragments, scored as identical, were not homologous. In all these cases, the differences occurred at the interspecific level. Our results suggest that RAPD data may be less reliable than RFLP data when estimating genetic relationships of accessions from more than one species.     

Genetic analysis of RFLPs, GATA microsatellites and RAPDs in a cross between L. esculentum and L. pimpinellifolium

A population of 257 BC₁ plants was developed from a cross between an elite processing line of tomato (Lycopersicon esculentum cvM82-1-7) and the closely related wild species L. pimpinellifolium (LA1589). The population was used to construct a genetic linkage map suitable for quantitative trait locus (QTL) analysis to be conducted in different backcross generations. The map comprises 115 RFLP, 3 RAPD and 2 morphological markers that span 1279 cM of the tomato genome with an average distance between markers of 10.7 cM. This map is comparable in length to that of the highdensity RFLP map derived from a L. esculentum x L. pennellii F₂ population. The order of the markers in the two maps is also in good agreement, however there are considerable differences in the distribution of recombination along the chromosomes. The segregation of six GATA-containing loci and 47 RAPD markers was also analyzed in subsets of the population. All of the microsatellite loci and 35 (75%) of the RAPDs mapped to clusters associated with centromeric regions.     

Efficiency of RFLP, RAPD, and AFLP markers for the construction of an intraspecific map of the tomato genome.

We have constructed a tomato genetic linkage map based on an intraspecific cross between two inbred lines of Lycopersicon esculentum and L. esculentum var. cerasiforme. The segregating population was composed of 153 recombinant inbred lines. This map is comprised of one morphological, 132 RFLP (restriction fragment length polymorphism, including 16 known-function genes), 33 RAPD (random amplified polymorphic DNA), and 211 AFLP (amplified fragment length polymorphism) loci. We compared the 3 types of markers for their polymorphism, segregation, and distribution over the genome. RFLP, RAPD, and AFLP methods revealed 8.7%, 15.8%, and 14.5% informative bands, respectively. This corresponded to polymorphism in 30% of RFLP probes, 32% of RAPD primers, and 100% of AFLP primer combinations. Less deviation from the 1:1 expected ratio was obtained with RFLP than with AFLP loci (8% and 18%, respectively). RAPD and AFLP markers were not randomly distributed over the genome. Most of them (60% and 80%, respectively) were grouped in clusters located around putative centromeric regions. This intraspecific map spans 965 cM with an average distance of 8.3 cM between markers (of the framework map). It was compared to other published interspecific maps of tomato. Despite the intraspecific origin of this map, it did not show any increase in length when compared to the high-density interspecific map of tomato.     

Integration of the classical and RFLP linkage maps of the short arm of tomato chromosome 1

The classical map of the short arm of chromosome 1 of tomato (Lycopersicon esculentum) has been shown to contain inaccuracies while the RFLP map of this region is known to be generally accurate. Molecular analysis of populations derived from crosses between L. esculentum lines carrying chromosome 1 classical markers and L. pennellii has enabled us to produce an integrated classical and RFLP marker map of this region. New data concerning the linkage relationships between classical markers have also been combined with previous data to produce a new classical map of the short arm of chromosome 1. The orders of the classical markers on these two new maps are in almost complete agreement and are very different to that shown on the previous classical map.     

Characterisation of a cytoplasmic male-sterile hybrid line between Lycopersicon peruvianum Mill. ×Lycopersicon pennellii Corr. and its crosses with cultivated tomato

 The cytoplasmic male-sterile (CMS) line CMS-pennellii (BC₁₀P₂ L. peruvianum×L. pennellii) and its complex hybrids with L. esculentum were studied. The established sterility was classified as the sporogenous type. As a result of the interaction of the genome of L. pennellii and the cytoplasm of L. peruvianum clear changes were established in the profiles of malic enzyme and esterase. Restriction fragment length polymorphism (RFLP) was detected between the mitochondrial (mt) genomes of CMS-pennellii and the cytoplasm donor, L. peruvianum, for two mtDNA probes: atpA and nad3. The established differences in the isozyme pattern and mt genomes are considered as useful markers to distinguish fertile and sterile plants. A breakthrough in the unilateral incompatibility of CMS-pennellii and the incorporation of the genome of L. esculentum on a CMS background is reported. The analysis of the complex hybrids assumes the interaction of two dominant genes – a maintainer gene from L. pennellii and a restorer gene from cultivated tomato. The hybrids produced with L. esculentum provide the basis for the development of a CMS system in cultivated tomato. Received: 25 May 1998 / Accepted: 26 August 1998     

A set of simple PCR markers converted from sequence specific RFLP markers on tomato chromosomes 9 to 12

A set of 24 simple PCR markers was generated for tomato chromosomes 9, 10, 11 and 12. Polymorphism was sought for between Lycopersicon esculentum and one of six other Lycopersicon species (L. parviflorum, L. cheesmanii, L. hirsutum, L. pennellii, L. peruvianum, and L. chilense). PCR primers, which were designed from mapped RFLP sequences, were used to amplify genomic DNA of the different species and the PCR amplification products were screened for polymorphism by testing restriction enzymes. With this approach, 24 (71%) of the 34 selected RFLPs were converted into simple PCR markers. By using a reference population, the map positions of these markers relative to the original RFLP markers were verified. These markers are locus specific and can be efficiently used for alignment of linkage maps, mapping target genes and marker assisted selection.     

Paracentromeric sequences on tomato chromosome 6 show homology to human satellite III and to the mammalian CENP-B binding box

In order to isolate centromeric sequences from tomato (Lycopersicon esculentum) chromosome 6, a large-scale RAPD screen was performed. Among 1500 polymorphic RAPD markers tested, 100 were identified as chromosome 6-specific, using a L. esculentum substitution line carrying chromosome 6 from L. pennellii. Fifty-seven of these markers proved to originate from L. pennellii, and of these, 13 were genetically mapped between the morphological markers yellow virescent (yv) and thiaminless (tl), which flank the centromere. These markers could be assigned to three genetic loci, with 11 of the markers mapping to a single locus. Further resolution of this cluster was achieved using radiation-induced deletions that removed yv or tl but not the centromere. Seven markers were shown to be located outside all of the deletions. These seven markers and three of the other markers of the cluster were cloned and sequenced. Five of the clones are present as single-copy or low-copy-number sequences and five represent middle repetitive sequences. Three sequences show homology to the mammalian CENP-B binding box; clone AG12 contains two of these boxes and also shows homology to human satellite III. Received: 13 January 1998 / Accepted: 21 April 1998     

Map-based cloning in crop plants. Tomato as a model system: I. Genetic and physical mapping of jointless

A map-based cloning scheme is being used to isolate the jointless (j) gene of tomato. The jointless locus is defined by a single recessive mutation that completely suppresses the formation of the fruit and flower pedicel and peduncle abscission zone. jointless was mapped in an F₂ population of an interspecific cross between Lycopersicon esculentum and Lycopersicon pennellii to a 7.1 cM interval between two restriction fragment length polymorphism (RFLP) markers TG523 and TG194. Isogenic DNA pools were then constructed from a subset of the mapping population and screened with 800 random decamers for random amplification of polymorphic DNA (RAPD) polymorphisms. Five new RAPD markers were isolated and mapped to chromosome 11, two of which were mapped within the targeted interval. One marker, RPD158, was mapped 1.5 cM to the opposite side of jointless relative to TG523 and thus narrowed the interval between the closest flanking markers to 3.0 cM. Physical mapping by pulse-field gel electrophoresis using TG523 and RPD158 as probes demonstrated that both markers hybridize to a common 600 kb SmaI restriction fragment. This provided an estimate of 200 kb/cM for the relationship between physical and genetic distances in the region of chromosome 11 containing the j locus. The combined results provide evidence for the feasibility of the next step toward isolation of the jointless gene by map-based cloning — a chromosome walk or jump to jointless.     

Resistance to powdery mildew (Oidium lycopersicum) in Lycopersicon hirsutum is controlled by an incompletely-dominant gene Ol-1 on chromosome 6

The inheritance of resistance to powdery mildew (Oidium lycopersicum) in Lycopersicon hirsutum was investigated by disease tests in segregating populations obtained by hybridising tomato (L. esculentum) cv Moneymaker with the wild relative L. hirsutum G1.1560. One incompletely dominant gene Ol-1 was found to largely control resistance to the disease. To map Ol-1, DNA pools from seven resistant and ten susceptible F₂ plants were analyzed for random amplified polymorphic DNA (RAPD). With 32 primers tested, one RAPD, primed with the sequence 5-GACGTGGTGA-3, was observed between the susceptible and the resistant bulks, which cosegregated with resistance in the F₂ population of L. esculentum × L. hirsutum G1.1560. This RAPD was mapped on chromosome 6 by using an F₂ (L. esculentum × L. pennellii) already mapped for 49 RFLPs. RFLP analysis of the F₂ from L. esculentum cv Moneymaker × L. hirsutum G1.1560 demonstrated that Ol-1 maps near the Aps-1 region on chromosome 6, in the vicinity of the resistance genes to Meloidogyne spp. (Mi) and to Cladosporium fulvum (Cf-2/Cf-5).     

Intra- and inter-specific variations in Lens revealed by RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were used to estimate intra- and interspecific variations in the genus Lens (lentil). Twenty cultivars of L. culinaris ssp. culinaris, including 11 microsperma (small-seeded) and nine macrosperma (large-seeded) types, and 16 wild relatives (four accessions each of L. culinaris ssp. orientalis, L. odemensis, L. nigricans and L. ervoides), were evaluated for genetic variability using a set of 40 random 10-mer primers. Fifty reproducibly scorable DNA bands were observed from ten of the primers, 90% of which were polymorphic. Genetic distances between each of the accessions were calculated from simple matching coefficients. A dendrogram showing genetic relationships between them was constructed by an unweighted pair-group method with arithmetical averages (UPGMA). This study revealed that (1) expect for L. ervoides, the level of intraspecific variation in cultivated lentil is lower than that in wild species, (2) L. culinaris ssp. orientalis is the most likely candidate for a progenitor of the cultivated species, and (3) microsperma and macrosperma cultivars were indistinguishable by the RAPD markers identified here.     

Lycopersicon esculentum lines containing small overlapping introgressions from L. pennellii

Summary The objective of this project was to introgress small overlapping chromosome segments which cover the genome of L. pennellii into Lycopersicon esculentum lines. The interspecific hybrid was backcrossed to L. esculentum, and a map of 981 cM, based on 146 molecular markers covering the entire genome, was produced. A similar backcross 1 population was selfed for six generations, under strong selection for cultivated tomato phenotypes, to produce 120 introgression lines. The introgression lines were assayed for the above-mentioned molecular markers, and 21 lines covering 936 cM of L. pennellii, with an average introgression of 86 cM, were selected to provide a resource for the mapping of new DNA clones. The rest of the lines have shorter introgressions consisting of specific regions with an average size of 38 cM. The proportion of the L. pennellii genome in the introgression lines was lower than expected (252 cM) because of strong selection against the wild-parent phenotype. The mean introgression rate for ends of linkage groups in the 120 lines was 3 times higher than for other regions of the genome. The introgression lines can assist in RFLP-based gene cloning by allowing the rapid selection of DNA markers that map to specific chromosome segments. The introgression lines also provide a base population for the mapping and breeding for quantitative traits such as salt and drought tolerance that characterize the wild species L. pennellii.     

Mapping the Sw-5 locus for tomato spotted wilt virus resistance in tomatoes using RAPD and RFLP analyses

The Sw-5 locus confers dominant resistance to tomato spotted wilt virus (TSWV). To map the location and facilitate the identification of markers linked to Sw-5 we developed a pair of near-isogenic lines (NILs) and an F₂ Lycopersicon esculentum x L. pennellii population segregating for resistance to TSWV. DNA from the NILs was analyzed using 748 random 10-mer oligonucleotides to discern linked molecular markers using a random amplified polymorphic DNA (RAPD) approach. One random primer (GAGCACGGGA) was found to produce a RAPD band of about 2200 bp that demonstrates linkage to Sw-5. Data from co-segregation of resistance and restriction fragment length polymorphisms (RFLPs) in a F₂ interspecific population position Sw-5 between the markers CT71 and CT220 near the telomere of the long arm of chromosome 9.     

Expression of unilateral incompatibility in pollen of Lycopersicon pennellii is determined by major loci on chromosomes 1, 6 and 10

Summary We have previously described gene introgression from the wild nightshade Solanum lycopersicoides into tomato (Lycopersicon esculentum) through the use of either diploid or sesquidiploid hybrids (the latter consisting of two genomes of L. esculentum and one genome of S. lycopersicoides). Both types of intergeneric hybrids display pollen sterility, but workable ovule fertility. Unilateral incompatibility prevents their direct hybridization with staminate L. esculentum. Pollen of a self-compattible form of the related wild species L. pennellii is compatible with pistils of L. esculentum x S. lycopersicoides hybrids. This trait was backcrossed from L. pennellii to L. esculentum in order to develop bridging lines that could be used to obtain progeny from the intergeneric hybrids and to study the inheritance of bridging ability. In progeny of L. esculentum x S. lycopersicoides hybrids pollinated with L. pennellii-derived bridging lines, preferential transmission of L. pennellii alleles was observed for certain isozyme and RFLP markers on chromosomes 1, 6 and 10. The skewed segregations suggest linkage to three major pollen-expressed compatibility loci. This was confirmed by observations of pollen tube growth, which indicated that compatibility with pistils of the diploid intergeneric hybrid occurred only in bridging lines at least heterozygous for the L. pennellii markers on chromosomes 1, 6 and 10. Compatibility with the sesquidiploid hybrid required only the chromosome 1 and 6 loci, indicating an apparent effect of gene dosage on expression of incompatibility in the pistil. In an F₂ L. esculentum x L. pennellii population, preferential transmission of L. pennellii alleles was observed for the same markers on chromosomes 1 and 10, as well as other markers on chromosomes 3, 11, and 12, but not 6. The chromosome 1 pollen compatibility locus maps to or near the S-locus, which determines S-allele specificity. The results are discussed in relation to existing genetic models for unilateral incompatibility, including the possible involvement of the S-locus.     

Molecular phylogeny in Indian Tinospora species by DNA based molecular markers

The phylogenetic relationships among the three species of Tinospora found in India are poorly understood. Morphology does not fully help to resolve the phylogeny and therefore a fast approach using molecular analysis was explored. Two molecular approaches viz Random Amplified Polymorphic DNA (RAPD) assay and restriction digestion of ITS1-5.8S-ITS2 rDNA (PCR-RFLP) were used to evaluate the genetic similarities between 40 different accessions belonging to three species. Of the 38 random primers used only six generated the polymorphism, while as three out of 11 restriction enzymes used gave polymorphic restriction patterns. The average proportion of polymorphic markers across primers was 95%, however restriction endonucleases showed 92% polymorphism. RAPD alone was found suitable for the species diversions. In contrast PCR- RFLP showed bias in detecting exact species variation. The correlation between the two markers was performed by Jaccard's coefficient of similarity. A significant (r= 0.574) but not very high correlation was obtained. Further to authenticate the results obtained by two markers, sequence analysis of ITS region of ribosomal DNA (ITS1 and ITS2, including 5.8S rDNA) was performed. Three independent clones of each species T. cordifolia, T. malabarica and T. crispa were sequenced. Phylogenetic relationship inferred from ITS sequences is in agreement with RAPD data.     

Molecular mapping of the py-1 gene for resistance to corky root rot (Pyrenochaeta lycopersici) in tomato

 We report the molecular mapping of the py-1 gene for resistance to corky root rot [Pyrenochaeta lycopersici (Schneider and Gerlach)] in tomato using RAPD and RFLP marker analysis. DNA from near-isogenic lines (NILs) of tomato differing in corky root rot resistance was screened with 575 random oligonucleotide primers to detect polymorphic DNAs linked to py-1. Three primers (OPW-04, OPC-02, OPG-19) revealed polymorphisms between the NILs. Twelve resistant and eight susceptible DNA pools derived from segregating F₃ families were used to confirm that the RAPD markers were linked to the py-1 gene. Two of the linked amplified fragments, corresponding to OPW-04 and OPC-02, were subsequently cloned and mapped on the tomato molecular linkage map as RFLPs. These clones were located between TG40 and CT31 on the short arm of chromosome 3. Further analysis with selected RFLP markers showed that 7% (8.8 cM) of chromosome 3 of the resistant line ‘Moboglan’ was introgressed from the L. peruvianum donor parent. Three RFLP markers (TG40, TG324, and TG479) from the introgressed part of chromosome 3 were converted to cleaved amplified polymorphism (CAP) markers for use in a polymerase chain reaction (PCR) assay. These PCR markers will allow rapid large-scale screening of tomato populations for corky root rot resistance. Received: 2 January 1998 / Accepted: 12 January 1998     

Mapping a new nematode resistance locus in Lycopersicon peruvianum

Accessions of the wild tomato species L. peruvianum were screened with a root-knot nematode population (557R) which infects tomato plants carrying the nematode resistance gene Mi. Several accessions were found to carry resistance to 557R. A L. peruvianum backcross population segregating for resistance to 557R was produced. The segregation ratio of resistant to susceptible plants suggested that a single, dominant gene was a major factor in the new resistance. This gene, which we have designated Mi-3, confers resistance against nematode strains that can infect plants carrying Mi. Mi-3, or a closely linked gene, also confers resistance to nematodes at 32°C, a temperature at which Mi is not effective. Bulked-segregant analysis with resistant and susceptible DNA pools was employed to identify RAPD markers linked to this gene. Five-hundred-and-twenty oligonucleotide primers were screened and two markers linked to the new resistance gene were identified. One of the linked markers (NR14) was mapped to chromosome 12 of tomato in an L. esculentum/L. pennellii mapping population. Linkage of NR14 and Mi-3 with RFLP markers known to map on the short arm of chromosome 12 was confirmed by Southern analysis in the population segregating for Mi-3. We have positioned Mi-3 near RFLP marker TG180 which maps to the telomeric region of the short arm of chromosome 12 in tomato.     

Relationships among cultivated and wild lentils revealed by RAPD analysis

RAPD markers were used to distinguish between six different Lens taxa, representing cultivated lentil and its wild relatives. Twenty-four arbitrary sequence 10-mer primers were identified which revealed robust and easily interpretable amplification-product profiles. These generated a total of 88 polymorphic bands in 54 accessions and were used to partition variation within and among Lens taxa. The data showed that, of the taxa examined, ssp. orientalis is most similar to cultivated lentil. L. ervoides was the most divergent wild taxon followed by L. nigricans. The genetic similarity between the latter two species was of the same magnitude as between ssp. orientalis and cultivated lentil. In addition, species-diagnostic amplification products specific to L. odemensis, L. ervoides and L. nigricans were identified. These results correspond well with previous isozyme and RFLP studies. RAPDs, however, appear to provide a greater degree of resolution at a sub-species level. The level of variation detected within cultivated lentils suggests that RAPD markers may be an appropriate technology for the construction of genetic linkage maps between closely related Lens accessions.On sabbatical leave from HP Agricultural University, Palampur 176 062, India     

DNA polymorphisms in grain sorghum (Sorghum bicolor (L.) Moench)

Molecular markers [random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP)] were used to determine the frequency of DNA polymorphism in grain sorghum (Sorghum bicolor (L.) Moench). Twenty-nine oligonucleotide primers were employed for RAPDs, generating a total of 262 DNA fragments, of which 145 were polymorphic in at least one pairwise comparison between 36 genotypes. Individual primers differed significantly in their ability to detect genetic polymorphism in the species. The overall frequency of polymorphisms was low with a mean frequency of 0.117 polymorphisms per RAPD band being obtained from all pairwise comparisons between genotypes, with maximum and minimum values of 0.212 and 0.039, respectively. Results from phenetic analysis of bandsharing data were consistent with current sub-specific groupings of the species, with clusters of Durra, Zerazera, Caud-Nig, Caud-Kaura and Caffrorum being discernible. The results also indicated that individuals of a similar taxonomic grouping but different geographic origin may be genetically less identical than previously considered. Similar frequencies of polymorphism to that obtained with RAPDs were obtained with RFLPs. Results from these experiments indicated that a high level of genetic uniformity exists within S. bicolor.     

Genetic variation in cultivated strains of <Emphasis Type="Italic">Agaricus blazei</Emphasis>

Genetic differences among Agaricus blazei strains were investigated using somatic incompatibility testing, isozyme analysis, restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA (mtDNA), and random amplified polymorphic DNA (RAPD) analysis. Eight strains, one cultivated strain from Brazil and seven from Japan, were used in this study. Somatic incompatibility interactions were observed between the Brazilian cultivated strain and the Japanese strains. The Brazilian cultivated strain had its own distinct patterns of esterase isozyme and mtDNA RFLP, but all seven Japanese cultivated strains showed identical patterns. When the RAPD patterns, obtained using eight primers, were compared the eight strains had their own distinct RAPD profiles. Distance values were calculated between all pairs of the strains based on presence or absence of individual RAPD bands, and a dendrogram was constructed by unweighted pair-group method with arithmetic clustering (UPGMA) analysis. Seven Japanese cultivated strains were grouped to each other, and this group was finally linked to the Brazilian cultivated strain. Based on these results, the degree of genetic variation among the A. blazei strains used is discussed.