RFLP markers linked to the root knot nematode resistance gene Mi in tomato

Summary The Mi gene originating from the wild tomato species Lycopersicon peruvianum confers resistance to all major root knot nematodes (Meloidogyne spp.). This single dominant gene is located on chromosome 6 and is very closely linked to the acid phosphatase-1 (Aps-1) locus. Resistance to nematodes has been introgressed into various cultivars of the cultivated tomato (L. esculentum), in many cultivars along with the linked L. peruvianum Aps-1 ¹ allele. By using a pair of nearly isogenic lines differing in a small chromosomal region containing the Mi and Aps-1 loci, we have identified two RFLP markers, GP79 and H6A2c2, which are located in the introgressed L. peruvianum region. Analysis of a test panel of 51 L. esculentum genotypes of various origins indicated that GP79 is very tightly linked to the Mi gene and allows both homozygous and heterozygous nematode-resistant genotypes to be distinguished from susceptible genotypes, irrespective of their Aps-1 alleles. Marker H6A2c2 is linked to the Aps-1 locus and is capable of discriminating between the L. peruvianum Aps-1 ¹ allele and the L. esculentum Aps-1 ³ and Aps-1 ⁺ alleles. In combination, these RFLP markers may provide a powerful tool in breeding tomatoes for nematode resistance.     

Identification of RFLP markers linked to the cereal cyst nematode resistance gene (Cre) in wheat

The cereal cyst nematode (CCN) (Heterodera avenae Woll.) is an economically damaging pest of wheat in many of the worlds cereal growing areas. The development of CCN-resistant cultivars may be accelerated by the use of molecular markers. The Cre gene of the wheat line AUS 10894 confers resistance to CCN. Using a pair of near-isogenic lines (NILs) that should differ only in a small chromosome segment containing the Cre locus, we screened 58 group-2 probes and found two (Tag605 and CDO588) that detect polymorphism between the NILs. Nulli-tetrasomic and ditelosomic lines confirmed that the restriction fragment length polymorphism (RFLP) markers identified were derived from the long arm of wheat chromosome 2. Crosses between AUS 10894 and Spear and the NIL AP and its recurrent parent Prins were used to produce F₂ populations that gave the expected 31 segregation ratio for the resistance gene. Linkage analysis identified two RFLP markers flanking the resistance gene. Xglk605 and Xcdo588 mapped 7.3 cM (LOD=6.0) and 8.4 cM (LOD=6.7), respectively, from the Cre locus.     

Identification of the double genome donor in spontaneous triploid tomato plants by RFLP analysis

Independent spontaneous triploid tomato plants (Lycopersicon esculentum Mill.) were collected among diploid hybrids growing in commercial greenhouses. Ploidy levels were verified by counting chromosomes, and the donor of the double genome dose was determined by restriction fragment length polymorphism (RFLP) analysis. The TG101 probe, which is tightly linked to the Tm-2 ^a locus, revealed different restriction patterns between TMV-resistant and TMV-susceptible parent lines. The parent donor which provided two genomes to the triploid was identified by comparing the relative intensity of alleles in the triploid with that in the diploid. The results indicate that both parents can serve as a double genome donor.     

RFLP linkage analysis of the Cf-4 and Cf-9 genes for resistance toCladosporium fulvum in tomato

Four different populations segregating for one of the two closely linked (possibly allelic) tomato disease resistance genes to the fungusCladosporium fulvum,Cf-4 andCf-9, were generated and analysed for recombination frequencies between theCf-genes and restriction fragment length polymorphism (RFLP) loci. The population consisting of F₂ progeny from the interspecific crossLycopersicon esculentum carryingCf-9 ×L. pennellii was identified as the most useful for RFLP mapping of theCf-4/9 locus and an RFLP map around this locus was constructed mainly using this population. The two closest markers identified were CP46, 2.6 cM distal, and a group of 11 markers including TG236, 3.7 cM proximal toCf-4/9. A polymerase chain reaction (PCR)-based procedure for the rapid identification of recombination events between these two markers was developed. The regions of foreign DNA introgression surroundingCf-4 andCf-9 in near-isogenic lines were delimited.     

A PCR-based marker tightly linked to the nematode resistance gene,Mi, in tomato

A PCR-based codominant marker has been developed which is tightly linked to Mi, a dominant genetic locus in tomato that confers resistance to several species of root-knot nematode. DNA from tomato lines differing in nematode resistance was screened for random amplified polymorphic DNA markers linked to Mi using decamer primers. Several markers were identified. One amplified product, REX-1, obtained using a pair of decamer primers, was present as a dominant marker in all nematode-resistant tomato lines tested. REX-1 was cloned and the DNA sequences of its ends were determined and used to develop 20-mer primers. PCR amplification with the 20-mer primers produced a single amplified band in both susceptible and resistant tomato lines. The amplified bands from susceptible and resistant lines were distinguishable after cleavage with the restriction enzyme Taq I. The linkage of REX-1 to Mi was verified in an F₂ population. This marker is more tightly linked to Mi than is Aps-1, the currently-used isozyme marker, and allows screening of germplasm where the linkage between Mi and Aps-1 has been lost. Homozygous and heterozygous individuals can be distinguished and the procedure can be used for rapid, routine screening. The strategy used to obtain REX-1 is applicable to obtaining tightly-linked markers to other genetic loci. Such markers would allow rapid, concurrent screening for the segregation of several loci of interest.     

Chloroplast DNA sequences integrated into an intron of a tomato nuclear gene

Summary DNA sequences capable of hybridizing with chloroplast DNA have previously been reported to exist in the nuclear genome of higher plants. Here we show that the third intron of the cultivated tomato (Lycopersicon esculentum) nuclear gene Cab-7, which resides on chromosome 10 and which we recently cloned and sequenced, contains two DNA fragments derived from the coding region of the chloroplast gene psbG. The first fragment, 133 bp long, is located at a site 63 bp from the 3 end of the 833 bp intron. The exact sequence of the 11 nucleotides at the 3 end of the inserting chloroplast sequence is also found at the 5 border of the insertion. A small (107 bp) chloroplast DNA fragment is inserted near the middle of the intron, again with the 3 end of the inserting element (6 bp) duplicated at the 5 border of the insertion. The second insert is a subfragment of the first insert, and is most likely directly derived from it. The psbG insertion sequence was found to be present in the Cab-7 gene of all tomato species examined but not in species from related genera (e.g. Solanum, Petunia, Nicotiana), suggesting that the original transposition event (chloroplast to nucleus) occurred relatively recently-since the divergence of the genus Lycopersicon from other genera in the family Solanaceae, but before radiation of species in that genus.     

Pulsed field gel electrophoresis and physical mapping of large DNA fragments in the Tm-2a region of chromosome 9 in tomato

Summary A method has been developed which allows the isolation of very high molecular weight DNA (>2 million bp) from leaf protoplasts of tomato (Lycopersicon esculentum). The DNA isolated in this manner was digested in agarose with rare-cutting restriction enzymes and separated by pulsed field gel electrophoresis. The size range of the reslting fragments was determined by hybridization to a number of single copy clones and the suitability of these enzymes for the mapping of large DNA fragments was evaluated. Furthermore, five genetically tightly linked single copy clones have been used to begin the construction of a physical map in a region of the genome containing the Tm-2a gene which confers resistance to tobacco mosaic virus. Two of the five clones were found to be on the same 560 kb SalI fragment and therefore are no further apart than that distance. The remaining three markers are distributed over at least 3 million bp, so that the total minimum physical distance of that cluster is at least 4 million bp. The results are discussed with respect to correlations between recombination frequencies and physical distance as well as physical mapping large regions of a complex plant genome like tomato.     

Molecular analysis of the extent of asymmetry in two asymmetric somatic hybrids of tomato

Summary Two somatic hybrid plants generated from a single fusion event between Lycopersicon esculentum and irradiated L. pennellii protoplasts have been analyzed at the molecular level. Over 30 loci have been analyzed using isozymes and RFLPs. All loci tested on chromosomes 2–10 were heterozygous, while those loci on chromosome 12 were homozygous L. pennellii in both somatic hybrids. In one of the somatic hybrids, 2850, loci on chromosome 1 were also homozygous L. pennellii. The other somatic hybrid, 28F5, was heterozygous at all chromosome 1 loci tested, but exhibited altered stoichiometry of parental bands as compared to the sexual hybrid. Loci on chromosome 2 from both somatic hybrids have altered stoichiometry, with L. pennellii alleles being four times more abundant than expected. Both somatic hybrids contain the L. esculentum chloroplast genome, while only L. pennellii polymorphisms have been detected in the mitochondrial genome.     

Fragments of plastid DNA in the nuclear genome of tomato: prevalence, chromosomal location, and possible mechanism of integration

Summary We have undertaken a systematic search for plastid DNA sequences integrated in the tomato nuclear genome, using heterologous probes taken from intervals of a plastid DNA region spanning 58 kb. A total of two short integrates (202 and 141 nucleotides) were isolated and mapped to chromosomes 9 and 5, respectively. The nucelotide sequence of the integrates and that of the flanking regions were determined. The integration sites contain direct repeat elements similar in position (but not in length or sequence) to the direct repeats previously observed with another plastid integrate in the tomato nuclear genome. Based on these results, a model for the process of movement and integration of plastid sequences into the nuclear genome is discussed.     

Expression of a magainin-type antimicrobial peptide gene (MSI-99) in tomato enhances resistance to bacterial speck disease

MSI-99 is a synthetic analog of magainin II (MII), a small cationic peptide highly inhibitory to a wide spectrum of microbial organisms. Tomato plants were transformed to express a gene encoding the MSI-99 peptide and tested for possible enhancement of resistance to important pathogens of this crop. Thirty-six tomato transformants carrying an MSI-99 expression vector designed to target the peptide into extracellular spaces were obtained by Agrobacterium tumefaciens-mediated transformation. Expression of MSI-99 caused no obvious cytotoxic effects in these plants. In the tests with Pseudomonas syringae pv. tomato (bacterial speck pathogen) at 10⁵ CFU/ml, several MSI-99-expressing lines developed significantly fewer disease symptoms than controls. However, MSI-99-expressing lines were not significantly different from controls in their responses to the fungal pathogen Alternaria solani (early blight) and the oomycete pathogen Phytophthora infestans (late blight). These findings are in accordance with our previous in vitro inhibition tests, which showed that the MSI-99 peptide is more inhibitory against bacteria than against fungi and oomycetes. Additional in vitro inhibition assays showed that MSI-99 loses its antimicrobial activity in the total or extracellular fluids from leaflets of non-transformed tomato plants; however, P. syringae pv. tomato could not multiply in the extracellular fluid from an MSI-99-expressing line. Our results suggest that expression strategies providing continuous high expression of MSI-99 will be necessary to achieve significant enhancement of plant disease resistance.Abbreviations AMP Antimicrobial peptide - CFU Colony forming unit - ECF Extracellular fluid - gus -glucuronidase gene - nptII Neomycin phosphotransferase II - SP Signal peptide - TF Total fluidCommunicated by S. Gleddie     

Molecular mapping of a major gene conferring resistance to maize mosaic virus

 The objective of this study was to determine the genetic basis of resistance to maize mosaic virus (MMV). Molecular markers were used to map resistance loci to MMV in a set of 91 maize (Zea mays L.) recombinant inbred lines (RILs), derived from the cross between Hi31 (a B68 conversion resistant to MMV) and Kil4 (a Thai inbred susceptible to MMV). The RILs were evaluated for MMV resistance in disease nurseries in Hawaii in the winter of 1993 and the summer of 1994. Twenty-eight highly susceptible RILs were chosen for gene mapping using the pooled-sampling approach. Initial evidence from the pooled DNA indicated that restriction fragment length polymorphism (RFLP) probes on chromosome 3 near the centromere were biased to the susceptible parent allele. Analysis of 91 RILs at 103 RFLP loci confirmed the presence of a major MMV resistance gene on chromosome 3. The resistant allele at this locus, previously named Mv1, is present in the resistant parent Hi31 and traces back to the Argentine parent used in conferring common rust resistance to B68. We conclude that resistance to MMV in B68 and Caribbean flints involves a major gene mv1 on chromosome 3 located between RFLP markers umc102 and php20508. Received: 12 June 1996 / Accepted: 5 July 1996     

Characterization of the level,target sites and inheritance of cytosine methylation in tomato nuclear DNA

The tomato nuclear genome was determined to have a G+C content of 37% which is among the lowest reported for any plant species. Non-coding regions have a G+C content even lower (32% average) whereas coding regions are considerably richer in G+C (46%).5-methyl cytosine was the only modified base detected and on average 23% of the cytosine residues are methylated. Immature tissues and protoplasts have significantly lower levels of cytosine methylation (average 20%) than mature tissues (average 25%). Mature pollen has an intermediate level of methylation (22%). Seeds gave the highest value (27%), suggesting de novo methylation after pollination and during seed development.Based on isoschizomer studies we estimate 55% of the CpG target sites (detected by Msp I/Hpa II) and 85% of the CpNpG target sites (detected by Bst NI/Eco RI)are methylated. Unmethylated target sites (both CpG and CpNpG) are not randomly distributed throughout the genome, but frequently occur in clusters. These clusters resemble CpG islands recently reported in maize and tobacco.The low G+C content and high levels of cytosine methylation in tomato may be due to previous transitions of 5mCT. This is supported by the fact that G+C levels are lowest in non-coding portions of the genome in which selection is relaxed and thus transitions are more likely to be tolerated. This hypothesis is also supported by the general deficiency of methylation target sites in the tomato genome, especially in non-coding regions.Using methylation isoschizomers and RFLP analysis we have also determined that polymorphism between plants, for cytosine methylation at allelic sites, is common in tomato. Comparing DNA from two tomato species, 20% of the polymorphisms detected by Bst NI/Eco RII could be attributed to differential methylation at the CpNpG target sites. With Msp I/Hpa II, 50% of the polymorphisms were attributable to methylation (CpG and CpNpG sites). Moreover, these polymorphisms were demonstrated to be inherited in a mendelian fashion and to co-segregate with the methylation target site and thus do not represent variation for transacting factors that might be involved in methylation of DNA. The potential role of heritable methylation polymorphism in evolution of gene regulation and in RFLP studies is discussed.     

Construction of an RFLP map of barley

Summary In order to construct an RFLP map of barley, two populations were analyzed using 251 genomic and cDNA markers: one population comprised 71 F₁ antherderived double haploid (DH) individuals of an intraspecific cross (IGRI x FRANKA), and the other 135 individuals of an interspecific F₂/F₃ progeny (VADA x H. spontaneum). The distribution of nonrepetitive clones over the seven barley chromosomes revealed a maximum for chromosome 2H and a minimum for 6H. The polymorphism of the interspecific progeny (76%) clearly exceeded that of the intraspecific progeny (26%) although, based on their pedigrees, IGRI and FRANKA are only distantly related. The contribution of individual chromosomes of the DH parents to the overall polymorphism varied between 8% and 50%. A significant portion (44% versus 10% of the interspecific progeny) of the markers mapped on the DH offspring showed distorted segregation, caused mainly by the prevalence of variants originating from the parent that better responded to in vitro culture (IGRI). In contrast to the interspecific map, probes displaying skewed segregation were clustered on the DH map on discrete segments. The colinear arrangement of both maps covers a distance of 1,453 cM and identifies regions of varying map distances.     

Agrobacterium-mediated transformation of cultivated tomato with construct carrying the nucleoprotein (N) gene from tomato spotted wilt virus (TSWV)

Significant yield losses in commercial tomato production caused by tomato spotted wilt virus (TSWV) are the reason why we have undertaken studies on resistance to this pathogen. One of the possible sources of resistance can be the incorporation of the nucleoprotein N viral gene by Agrobacterium transformation. The N gene was introduced into three Lycopersicon esculentum forms. Out of the total of 3044 cotyledon explants 14.7% regenerated shoots, but only a few were rooted on medium containing kanamycin. The preliminary analysis indicated that 18 plants are putative transformants.     

RFLP inheritance and linkage in walnut

Thirty-two low-copy-number genomic DNA clones from a walnut (Juglans sp.) Pst I genomic library were used to establish a molecular-marker linkage map for walnut. The clones were hybridized to restriction-endonuclease-digested DNA from parent walnut trees involved in an interspecific backcross of (J. hindsii x J. regia) x J. regia in order to identify parental polymorphism. Sixty-three backcross progeny were analyzed to determine the inheritance and linkage of 48 RFLP loci. Sixty-six percent of the walnut cloned sequences detected duplicated, but unlinked, loci. Twelve linkage groups were identified by 42 of the RFLP loci. A Poisson probability method for estimating genome size was utilized to calculate the approximate walnut genome length as 1660 cM and to estimate that 138 markers would be needed to cover 95% of the walnut genome within 20 cM of each marker.     

Tagging genes for blast resistance in rice via linkage to RFLP markers

Summary Both Pi-2(t) and Pi-4(t) genes of rice confer complete resistance to the blast fungal pathogen Pyricularia oryzae Cav. As economically important plant genes, they have been recently characterized phenotypically, yet nothing is known about their classical linkage associations and gene products. We report here the isolation of DNA markers closely linked to these blast resistance genes in rice. The DNA markers were identified by testing 142 mapped rice genomic clones as hybridization probes against Southern blots, consisting of DNA from pairs of nearly isogenic lines (NILs) with or without the target genes. Chromosomal segments introgressed from donor genomes were distinguished by restriction fragment length polymorphisms (RFLPs) between the NILs. Linkage associations of the clones with Pi-2(t) and Pi4(t) were verified using F₃ segregating populations of known blast reaction. Cosegregation of the resistant genotype and donor-derived allele indicated the presence of linkage between the DNA marker and a blast resistance gene. RFLP analysis showed that Pi-2(t) is closely linked to a single-copy DNA clone RG64 on chromosome 6, with a distance of 2.8+1.4(SE) cMorgans. Another blast resistance gene, Pi-4(t), is 15.3+4.2(SE) cMorgans away from a DNA clone RG869 on chromosome 12. These chromosomal regions can now be examined with additional markers to define the precise locations of Pi-2(t) and Pi-4(t). Tightly linked DNA markers may facilitate early selection for blast resistance genes in breeding programs. These markers may also be useful to map new genes for resistance to blast isolates. They may ultimately lead to the cloning of those genes via chromosome walking. The gene tagging approach demonstrated in this paper may apply to other genes of interest for both monogenic and polygenic traits.     

RFLP mapping of the ym4 virus resistance gene in barley

RFLP (restriction fragment length polymorphism) mapping of a recessive gene (ym4) conferring resistance to barley yellow mosaic and barley mild mosaic virus was performed using progeny of 86 F₁ anther-derived doubled haploid lines. Two closely linked RFLP markers that flank the gene at a distance of 1.2 centiMorgans were identified. Using one of these markers (MWG10) we obtained a clear differentiation between resistant and susceptible German cultivars. An analysis of a series of unrelated barley lines with probe MWG10 did not reveal additional RFLP fragments. The use of this probe for both marker-assisted selection and the generation of a high-density map around the resistance locus is discussed.     

Development of an RFLP linkage map in diploid peanut species

An RFLP linkage map of peanut has been developed for use in genetic studies and breeding programs aimed at improving the cultivated species (Arachis hypogaea L.). An F₂ population derived from the interspecific hybridization of two related diploid species in the sectionArachis (A. stenosperma ×A. cardenasii) was used to construct the map. Both random genomic and cDNA clones were used to develop the framework of the map. In addition, three cDNA clones representing genes coding for enzymes involved in the lipid biosynthesis pathway have been mapped in peanut. Of the 100 genomic and 300 cDNA clones evaluated, 15 and 190, respectively, revealed polymorphisms among the parents of our mapping population. Unfortunately, a large number of these produced complex banding patterns that could not be mapped. Of the 132 markers analyzed for segregation, 117 are distributed among 11 linkage groups, while 15 have not yet been associated with any other marker. A total map distance of approximately 1063 cM has been covered to-date.     

RFLP mapping of a major bruchid resistance gene in mungbean (Vigna radiata,L. Wilczek)

Summary Bruchids (genus Callosobruchus) are among the most destructive insect pests of mungbeans and other members of the genus, Vigna. Genetic resistance to bruchids was previously identified in a wild mungbean relative, TC1966. To analyze the underlying genetics, accelerate breeding, and provide a basis for map-based cloning of this gene, we have mapped the TC1966 bruchid resistance gene using restriction fragment length polymorphism (RFLP) markers. Fifty-eight F₂ progeny from a cross between TC1966 and a susceptible mungbean cultivar were analyzed with 153 RFLP markers. Resistance mapped to a single locus on linkage group VIII, approximately 3.6 centimorgans from the nearest RFLP marker. Because the genome of mungbean is relatively small (estimated to be between 470 and 560 million base pairs), this RFLP marker may be suitable as a starting point for chromosome walking. Based on RFLP analysis, an individual was also identified in the F₂ population that retained the bruchid resistance gene within a tightly linked double crossover. This individual will be valuable in developing resistant mungbean lines free of linkage drag.