Genetic and physical mapping of the patatin genes in potato and tomato

Summary Genes for the major storage protein of potato, patatin, have been mapped genetically and physically in both the potato and tomato genomes. In potato, all patatin genes detected by the cDNA clone pGM01 map to a single locus at the end of the long arm of chromosome 8. By means of pulsed field gel electrophoresis (PFGE) it was possible further to delimit this locus, containing 10–15 copies of the gene, to a maximum size of 1.4 million base pairs. Hybridizations with class-specific clones suggest that the locus is at least partially divided into domains containing the two major types of patatin genes, class I and II. In tomato, patatin-homologous sequences were found to reside at the orthologous locus at the end of chromosome 8. The approximately three copies in tomato were localized by PFGE to a single fragment of 300 kilobases. Whereas the class II-specific 5 promoter sequences reside in tomato at the same locus as the coding sequences, the single class I-specific copy of the 5 promoter sequences was localized on chromosome 3 with no coding sequence attached to it. A clone from this chromosome 3 locus of tomato was isolated and by restriction fragment length polymorphism mapping it could be further shown that a similar class I-specific sequence also exists on chromosome 3 of potato. As in tomato, this copy on chromosome 3 is not linked to a coding sequence for patatin. The results are discussed with respect to genome evolution and PFGE analysis of complex gene families.     

Construction of a yeast artificial chromosome library of tomato and identification of cloned segments linked to two disease resistance loci

Summary We have constructed a yeast artificial chromosome (YAC) library of tomato for chromosome walking that contains the equivalent of three haploid genomes (22 000 clones). The source of high molecular weight DNA was leaf protoplasts from the tomato cultivars VFNT cherry and Rio Grande-PtoR, which together contain loci encoding resistance to six pathogens of tomato. Approximately 11 000 YACs have been screened with RFLP markers that cosegregate withTm-2a andPto — loci conferring resistance to tobacco mosaic virus andPseudomonas syringae pv.tomato, respectively. Five YACs were identified that hybridized to the markers and are therefore starting points for chromosome walks to these genes. A subset of the library was characterized for the presence of various repetitive sequences and YACs were identified that carried TGRI, a repeat clustered near the telomeres of most tomato chromosomes, TGRII, an interspersed repeat, and TGRIIl, a repeat that occurs primarily at centromeric sites. Evaluation of the library for organellar sequences revealed that approximately 10% of the clones contain chloroplast sequences. Many of these YAC clones appear to contain the entire 155 kb tomato chloroplast genome. The tomato cultivars used in the library construction, in addition to carrying various disease resistance genes, also contain the wild-type alleles corresponding to most recessive mutations that have been mapped by classical linkage analysis. Thus, in addition to its utility for physical mapping and genome studies, this library should be useful for chromosome walking to genes corresponding to virtually any phenotype that can be scored in a segregating population.     

Characterization and genetic mapping of a short, highly repeated, interspersed DNA sequence from rice (Oryza sativa L.).

Summary A short, highly repeated, interspersed DNA sequence from rice was characterized using a combination of techniques and genetically mapped to rice chromosomes by restriction fragment length polymorphism (RFLP) analysis. A consensus sequence (GGC)_n, where n varies from 13–16, for the repeated sequence family was deduced from sequence analysis. Southern blot analysis, restriction mapping of repeat element-containing genomic clones, and DNA sequence analysis indicated that the repeated sequence is interspersed in the rice genome, and is heterogeneous and divergent. About 200000 copies are present in the rice genome. Single copy sequences flanking the repeat element were used as RFLP markers to map individual repeat elements. Eleven such repeat elements were mapped to seven different chromosomes. The strategy for characterization of highly dispersed repeated DNA and its uses in genetic mapping, DNA fingerprinting, and evolutionary studies are discussed.     

Restriction fragment length polymorphisms in mitochondrial DNA and ribosomal RNA gene complexes as an aid to the characterization of species and sub-species populations in the genus Verticillium

Abstract Genomic DNA was extracted from seven species of Verticillium and digested with the restriction endonucleases Eco RI or Hae III. Hybridization with an homologous V. albo-atrum ribosomal RNA gene probe revealed restriction fragment length polymorphisms (RFLPs) which could differentiate V. lateritium, V. lecanii, V. nigrescens, V. nubilum and V. tricorpus . Digestion with Eco RI did not provide RFLPs which could distinguish between V. albo-atrum and V. dahliae . Digestion of genomic and mitochondrial DNA with Hae III showed distinctive patterns on ethidium bromide gels which allowed each species to be distinguished. Some intra-species variation in patterns occurred and a combination of mitochondrial and ribosomal RNA gene complex RFLPs has potential as an aid for the characterization of species and sub-species populations in the genes Verticillium .     

Molecular cloning and sequence analysis of the lysR gene from the extremely thermophilic eubacterium, Thermus thermophilus HB27

We have isolated a lysine-auxotrophic and kanamycin-resistant mutant from an extreme thermophile, Thermus thermophilus HB27. This mutant showed the lysA⁻ or lysR⁻ genotype since it could not grow on the minimal plate which contained diaminopimelic acid. Sequence analysis of the clones which could rescue the Lys⁻ mutant indicated the lysR gene. The lysR gene overlapped with the rimK gene for the modification enzyme of ribosomal protein S6. In the Lys⁻ mutant, the lysR gene was disrupted and the C-terminus region of the RimK protein was different from that of the wild-type, which contributed to the Lys⁻ and kanamycin-resistant phenotype. The deduced amino acid sequence of the lysR gene showed 20.9% identity with the LysR protein of Escherichia coli. The percentage of use of cytosine or guanine in the third letter of the codons in the lysR gene was only 67.4%. We also determined that the argC gene encoding N-acetyl-γ-glutamyl phosphate reductase and the argB gene encoding acetylglutamate kinase were located immediately upstream of the lysR gene.     

Variability and genetics of spacer DNA sequences between the ribosomal-RNA genes of hexaploid wheat (Triticum aestivum)

Summary Using restriction enzyme digests of genomic DNA extracted from the leaves of 25 hexaploid wheat (Triticum aestivum L. em. Thell.) cultivars and their hybrids, restriction fragment length polymorphisms of the spacer DNA which separates the ribosomal-RNA genes have been examined. (From one to three thousand of these genes are borne on chromosomes 1B and 6B of hexaploid wheat). The data show that there are three distinct alleles of the 1B locus, designated Nor-B1a, Nor-B1b, and Nor-B1c, and at least five allelic variants of the 6B locus, designated Nor-B2a, Nor-B2b, Nor-B2c, Nor-B2d, and Nor-B2e. A further, previously reported allele on 6B has been named Nor-B2f. Chromosome 5D has only one allelic variant, Nor-D3. Whereas the major spacer variants of the 1B alleles apparently differ by the loss or gain of one or two of the 133 bp sub-repeat units within the spacer DNA, the 6B allelic variants show major differences in their compositions and lengths. This may be related to the greater number of rDNA repeat units at this locus. The practical implications of these differences and their application to wheat breeding are discussed.     

Primers for sequence characterization and polymorphism detection in the Atlantic salmon (Salmo salar) growth hormone 1 (GH1) gene

We report the identification of intraspecific sequence variation in the Atlantic salmon (Salmo salar) growth hormone 1 gene. Rapid and inexpensive assays for polymorphism detection were developed for 10 sites. Five of the assays detected single nucleotide polymorphisms (SNPs) using polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analyses, and five were indel polymorphisms, detected using fragment length analyses. The average within population frequency of the most common allele varied from 0.52 to 0.90, and the average within population heterozygosity varied from 0.02 to 0.37 in seven European salmon populations.     

Assessment of the degree and the type of restriction fragment length polymorphism in barley (Hordeum vulgare)

Summary In order to determine the extent of polymorphism in barley (Hordeum vulgare), DNA from 48 varieties was analyzed with 23 genomic, single-copy probes, distributed across all seven chromosomes. Upon hybridization to wheat-barley addition lines, the probes showed different degrees of homology compared to the wheat genome. Polymorphisms were detected in the barley genome at a frequency of 43% after digestion with EcoRI, BamHI, and HindIII. Subgroups of spring and winter barley and of two- and six-rowed types showed less diversity which, in most cases, was due to shifts in allelic frequencies. One probe (MWG1H504) hybridized to an EcoRI restriction fragment exclusively observed in winter barley. A comparison of six different restriction enzymes revealed clear differences with regard to their efficiency in detecting polymorphisms. The respective frequencies were between 13% (HindIII) and 37% (EcoRV). A significant correlation between the efficiency of a restriction enzyme and the mean fragment size detected by the different probes identified insertion/deletion events as the major factor causing polymorphism in barley.     

Comparative genome analysis of mungbean (Vigna radiata L. Wilczek) and cowpea (V. unguiculata L. Walpers) using RFLP mapping data

Genome relationships between mungbean (Vigna tradiata) and cowpea (V. Unguiculata) based on the linkage arrangement of random genomic restriction fragment length polymorphism (RFLP) markers have been investigated. A common set of probes derived from cowpea, common bean (Phaseolus vulgaris), mungbean, and soybean (Glycine max) PstI genomic libraries were used to construct the genetic linkage maps. In both species, a single F₂ population from a cross between an improved cultivar and a putative wild progenitor species was used to follow the segregation of the RFLP markers. Approximately 90% of the probes hybridized to both mungbean and cowpea DNA, indicating a high degree of similarity in the nucleotide sequences among these species. A higher level of polymorphism was detected in the mungbean population (75.7%) than in the cowpea population (41.2%). Loci exhibiting duplications, null phenotypes, and distorted segregation ratios were detected in both populations. Random genomic DNA RFLP loci account for about 89% of the currently mapped markers with a few cDNA and RAPD markers added. The current mungbean map is comprised of 171 loci/loci clusters distributed in 14 linkage groups spanning a total of 1570cM. On the other hand, 97 markers covered 684 cM and defined 10 linkage groups in the current cowpea map. The mungbean and cowpea genomes were compared on the basis of the copy number and linkage arrangement of 53 markers mapped in common between the two species. Results indicate that nucleotide sequences are conserved, but variation in copy number were detected and several rearrangements in linkage orders appeared to have occurred since the divergence of the two species. Entire linkage groups were not conserved, but several large linkage blocks were maintained in both genomes.     

Isolation and identification of a non-specific tandemly repeated DNA sequence in Oryza species

A tandemly repeated DNA sequence (RRS7) was isolated from Oryza alta (CCDD). RRS7-related sequences were also found tandemly arrayed in genomes AA, BB, BBCC, CC, and EE, and a small amount of RRS7-related sequences were detected in genome FF and the Oryza species with unknown genomes. DNA sequence analysis of the 1844-bp insert of RRS7 revealed that it contained six tandemly repeated units, of which five were 155 bp in length and one was 194 bp in length and contained an imperfect internal 39-bp duplication. Southern blot analysis showed that the boundary sequence contained in RRS7 is a single-copy sequence. A 155-bp consensus sequence derived from the six monomeric repeats contained no internal repeat and showed no significant homology to other currently known sequences. The results of Southern blot and sequence analysis revealed that there are at least two subfamilies present in the RRS7 family; these are represented by the DraI site and the MspI site, respectively. Restriction digestion with two pairs of isoschizomers MboI/Sau3A and MspI/HpaII demonstrated that most of the C residues in the GATC sites and the internal C in the CCGG sites of the RRS7 family in O. Alta were methylated. The usefulness of the RRS7 family in determining the evolutionary relationship of the genome DD and other Oryza genomes is discussed.     

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.     

Genetic localization of a bruchid resistance gene and its relationship to insecticidal cyclopeptide alkaloids, the vignatic acids, in mungbean ( Vigna radiata L. Wilczek)

Bruchid resistance, controlled by a single dominant gene (Br) in a wild mungbean accession (TC1966), has been incorporated into cultivated mungbean (Vigna radiata). The resistance gene simultaneously confers inhibitory activity against the bean bug, Riptortus clavatus Thunberg (Hemiptera: Alydidae). The resultant isogenic line (BC₂₀ generation) was characterized by the presence of a group of novel cyclopeptide alkaloids, called vignatic acids. A linkage map was constructed for Br and the vignatic acid gene (Va) using restriction fragment length polymorphism (RFLP) markers and a segregating BC₂₀F₂ population. By screening resistant and susceptible parental lines with 479 primers, eight randomly amplified polymorphic DNA (RAPD) markers linked to Br were identified and cloned for use as RFLP probes. All eight RAPD-based markers, one mungbean, and four common bean genomic clones were effectively integrated around Br within a 3.7-cM interval. Br was mapped to a 0.7-cM segment between a cluster consisting of six markers and a common bean RFLP marker, Bng110. The six markers are closest to the bruchid resistance gene, approximately 0.2 cM away. The vignatic acid gene, Va, cosegregated with bruchid resistance. However, one individual was identified in the BC₂₀F₂ population that retained vignatic acids in spite of its bruchid susceptibility. Consequently, Va was mapped to a single locus at the same position as the cluster of markers and 0.2 cM away from Br. These results suggest that the vignatic acids are not the principal factors responsible for bruchid resistance in V. radiata but will facilitate the use of map-based cloning strategies to isolate the Br gene. Received: 20 November 1997 / Accepted: 6 January 1998     

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.     

Ribosomal DNA as a convenient probe to follow segregation and possible divergency from expected homozygosity after haploidization of an androgenetic process

Summary Restriction fragment length polymorphism of the wheat nuclear ribosomal DNA has been studied in several steps of a breeding scheme, including parental genotypes, F1 hybrid, F9 generation, and anther-derived doubled haploid lines obtained from F9. Ribosomal DNA represents a suitable molecular marker in following segregation and possible divergency from expected homozygosity after haploidization of an androgenetic process. It has been shown to undergo variations among the first cycle-doubled haploid lines in the relative amount of two different sizes of ribosomal DNA repeat units. The specificity and peculiar properties of the plant system used allowed us to assign an intrachromosomal location (short arm of the chromosomes 1B, 1R or 6B) to several ribosomal DNA repeat units that differ by the length of their nontranscribed spacer region.     

RFLP tagging of a new semidwarfing gene in rice

A new rice semidwarfing gene which is not allelic tosd1, temporarily designated assdg, might be of use as a new source of semidwarfism in rice breeding programs. We report here the identification of a DNA marker closely linked to this gene. The DNA marker was identified by testing 120 mapped rice RFLP makers as hybridization probes for Southern analysis of a pair of nearly isogenic lines with or withoutsdg. Linkage association of the marker with the gene was verified using a F₂ population segregating for semidwarfism. RFLP analysis showed thatsdg is closely linked to a single-copy DNA clone RZ182 on chromosome 5, with a distance of 4.3 centiMorgans between them. This marker may facilitate early selection for the semidwarfing gene in rice breeding programs     

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.     

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.     

Molecular markers (RFLPs and HSPs) for the genetic dissection of thermotolerance in maize

Summary Cellular membrane stability (CMS) is a physiological index widely used to evaluate thermostability in plants. The genetic basis of the character has been studied following two different approaches: restriction fragment length polymorphism (RFLP) analysis, and the effects of segregating heat shock protein (HSP) loci. RFLP analysis was based on a set of recombinant inbreds derived from the T32 × CM37 F₁ hybrid and characterized for about 200 RFLP loci. Heritability of CMS estimated by standard quantitative analysis was 0.73. Regression analysis of CMS on RFLPs detected a minimum number of six quantitative trait loci (QTL) accounting for 53% of the genetic variability. The analysis of the matrices of correlation between RFLP loci, either within or between chromosomes, indicates that no false assignment was produced by this analysis. The effect of HSPs on the variability of the CMS was tested for a low-molecular-weight peptide (HSP-17) showing presence-absence of segregation in the B73 × Pa33 F₂ population. Although the genetic variability of the character was very high (h ²=0.58) the effect of HSP-17 was not significant, indicating either that the polypeptide is not involved in the determination of the character or that its effect is not statistically detectable.     

Integrated map of AFLP, SSLP and RFLP markers using a recombinant inbred population of rice (Oryza sativa L.)

 A molecular map of rice consisting of 231 amplified fragment length polymorphisms (AFLPs), 212 restriction fragment length polymorphisms (RFLPs), 86 simple-sequence length polymorphisms (SSLPs), five isozyme loci, and two morphological mutant loci [phenol staining of grain (Ph), semi-dwarf habit (sd-1)] has been constructed using an F₁₁ recombinant inbred (RI) population. The mapping population consisted of 164 RI lines and was developed via single-seed descent from an intercross between the genetically divergent parents Milyang 23 (M) (tongil type) and Gihobyeo (G) ( japonica type). A subset of previously mapped RFLP and SSLP markers were used to construct the map framework. The AFLP markers were derived from ten EcoRI(+2) and MseI(+3) primer combinations. All marker types were well distributed throughout the 12 chromosomes. The integrated map covered 1814 cM, with an average interval size of 3.4 cM. The MG map is a cornerstone of the Korean Rice Genome Research Program (KRGRP) and is being continuously refined through the addition of partially sequenced cDNA markers derived from an immature-seed cDNA library developed in Korea, and microsatellite markers developed at Cornell. The population is also being used for quantitative trait locus (QTL) analysis and as the basis for marker-assisted variety development. Received: 24 June 1997 / Accepted: 25 November 1997     

Differences between,and possible origins of,the cytoplasms found in fertile and male-sterile onions (Allium cepa L.)

Summary The DNA of the organellar genomes of Allium cepa has been examined to detect restriction fragment length polymorphisms. Differences can be shown between both the chloroplastal and mitochondrial genomes of the N and cms-S cytoplasms in their restriction fragment profiles. Southern blot analysis of the mtDNA profiles using probes containing defined mitochondrial genes also detected polymorphisms. No differences can be shown between the organellar genomes of the N and cms-T onions by either of these techniques. These data indicate different origins for the two sterility-conferring cytoplasms, suggesting autoplasmic and alloplasmic origins for the cms-T and cms-S cytoplasms, respectively. No evidence of the presence of virus-like particles was found in any of the cytoplasms.