Tomato genome is comprised largely of fast-evolving, low copy-number sequences

Summary Fifty random clones (350–2300 bp), derived from sheared, nuclear DNA, were studied via Southern analysis in order to make deductions about the organization and evolution of the tomato genome. Thirty-four of the clones were mapped genetically and determined to represent points on 11 of the 12 tomato chromosomes. Under moderate stringency conditions (80% homology required) 44% of the clones were classified as single copy. Under higher stringency, the majority of the clones (78%) behaved as single copy. Most of the remaining clones belonged to multicopy families containing 2–20 copies, while a few contained moderately or highly repeated sequences (10% at moderate stringency, 4% at high stringency). Divergence rates of sequences homologous to the 50 random genomic clones were compared with those corresponding to 20 previously described cDNA (coding sequence) clones. Rates were measured by probing each clone (random genomics and cDNAs) onto filters containing DNA from various species from the family Solanaceae (including potato, Datura, petunia and tobacco) as well as one species (watermelon) from another plant family, Cucurbitaceae. Under moderate stringency conditions, the majority of the random clones (single copy and repetitive) failed to detect homologous sequences in the more distantly related species, whereas approximately 90% of the 20 coding sequences analyzed could still be detected in all solanaceous species. The most highly repeated sequences appear to be the fastest evolving and homologous copies could be detected only in species most closely related to tomato. Dispersion of repetitive sequences, as opposed to tandem clustering, appears to be the rule for the tomato genome. None of the repetitive sequences discovered by this random sampling of the genome were tandemly arranged — a finding consistent with the notion that the tomato genome contains only a small fraction of satellite DNA. This study, along with a companion paper (Ganal et al. 1988), provides the first general sketch of the tomato genome at the molecular level and indicates that it is comprised largely of single copy sequences and these sequences, together with repetitive sequences are evolving at a rate faster than the coding portion of the genome. The small genome and paucity of highly repetitive DNA are favourable attributes with respect to the possibilities of conducting chromosome walking experiments in tomato and the fact that coding regions are well conserved among solanaceous species may be useful for distinguishing clones that contain coding regions from those that do not.     

Genetics of actin-related sequences in tomato

Summary The genomic distribution of actin-related sequences in tomato was investigated using a cloned actin gene from soybean. Ten actin loci account for most of the hybridizing fragments observed with Southern analysis. Single loci were found on chromosomes 1, 3 and 10 and two loci on chromosome 4. One locus is linked to an unmapped isozyme marker, Sod-1. The four remaining actin loci are independent of each other and of any of the other markers tested. The number of actin loci in tomato (10) is greater than that estimated for soybean (8). As soybean is apparently a tetraploid and tomato a diploid, these results suggest that the number of actin loci has not been stable during the evolution of dicots. A number of these mapped loci lie in regions of the genome previously devoid of molecular markers and thus may be useful in basic and applied genetic research.     

Copy number lability and evolutionary dynamics of the Adh gene family in diploid and tetraploid cotton (Gossypium)

Nuclear-encoded genes exist in families of various sizes. To further our understanding of the evolutionary dynamics of nuclear gene families we present a characterization of the structure and evolution of the alcohol dehydrogenase (Adh) gene family in diploid and tetraploid members of the cotton genus (Gossypium, Malvaceae). A PCR-based approach was employed to isolate and sequence multiple Adh gene family members, and Southern hybridization analyses were used to document variation in gene copy number. Adh gene copy number varies among Gossypium species, with diploids containing at least seven Adh loci in two primary gene lineages. Allotetraploid Gossypium species are inferred to contain at least 14 loci. Intron lengths vary markedly between loci, and one locus has lost two introns usually found in other plant Adh genes. Multiple examples of apparent gene duplication events were observed and at least one case of pseudogenization and one case of gene elimination were also found. Thus, Adh gene family structure is dynamic within this single plant genus. Evolutionary rate estimates differ between loci and in some cases between organismal lineages at the same locus. We suggest that dynamic fluctuation in copy number will prove common for nuclear genes, and we discuss the implications of this perspective for inferences of orthology and functional evolution.     

A low-density genetic map of onion reveals a role for tandem duplication in the evolution of an extremely large diploid genome

 The bulb onion, Allium cepa L., is a diploid (2n=2x=16) plant with a huge nuclear genome. Previous genetic and cytogenetic analyses have not supported a polyploid origin for onion. We developed a low-density genetic map of morphological markers, randomly amplified polymorphic DNAs (RAPD), and restriction fragment length polymorphisms (RFLP) as a tool for onion improvement and to study the genome organization of onion. A mapping population of 58 F₃ families was produced from a single F₁ plant from the cross of two partially inbred lines (Brigham Yellow Globe 15-23 and Alisa Craig 43). Segregations were established for restoration of male fertility in sterile cytoplasm, complementary light-red bulb color, 14 RAPDs, 110 RFLPs revealed by 90 anonymous cDNA clones, and 2 RFLPs revealed by a cDNA clone of alliinase, the enzyme responsible for the characteristic Allium flavors. Duplicated RFLP loci were detected by 21% of the clones, of which 53% were unlinked (>30 cM), 5% loosely linked (10–30 cM), and 42% tightly linked (<10 cM). This duplication frequency is less than that reported for paleopolyploids but higher than for diploid species. We observed 40% dominant RFLPs, the highest yet reported among plants. Among duplicated RFLP loci, 19% segregated as two loci each with two codominant alleles, 52% segregated as one locus with codominant alleles and one locus with only a dominant fragment, and 29% segregated as two loci with only dominant fragments. We sequenced cDNAs detecting duplicated RFLPs; 63% showed homology to known gene families (e.g., chlorophyll binding proteins, ubiquitin, or RuBISCO), and 37% were unique clones showing significant homology to known genes of low-copy number or no homology to database sequences. Duplicated RFLPs showing linkage could be due to retroviral-like sequences in adjacent coding regions or intrachromosomal, as opposed to whole genome, duplications. Previous cytological analyses and this genetic map support intrachromosomal duplication as a mechanism contributing to the huge onion genome. Received: 3 July 1997 / Accepted: 8 August 1997     

Mapping of RFLP and qualitative trait loci in Brassica rapa and comparison to the linkage maps of B. napus,B. oleracea,and Arabidopsis thaliana

A linkage map of restriction fragment length polymorphisms (RFLPs) was constructed for oilseed, Brassica rapa, using anonymous genomic DNA and cDNA clones from Brassica and cloned genes from the crucifer Arabidopsis thaliana. We also mapped genes controlling the simply inherited traits, yellow seeds, low seed erucic acid, and pubescence. The map included 139 RFLP loci organized into ten linkage groups (LGs) and one small group covering 1785 cM. Each of the three traits mapped to a single locus on three different LGs. Many of the RFLP loci were detected with the same set of probes used to construct maps in the diploid B. oleracea and the amphidiploid B. napus. Comparisons of the linkage arrangements between the diploid species B. rapa and B. oleracea revealed six LGs with at least two loci in common. Nine of the B. rapa LGs had conserved linkage arrangements with B. napus LGs. The majority of loci in common were in the same order among the three species, although the distances between loci were largest on the B. rapa map. We also compared the genome organization between B. rapa and A. thaliana using RFLP loci detected with 12 cloned genes in the two species and found some evidence for a conservation of the linkage arrangements. This B. rapa map will be used to test for associations between segregation of RFLPs, detected by cloned genes of known function, and traits of interest.     

The nematode-resistance gene,<Emphasis Type="Italic"> Mi-1</Emphasis>, is associated with an inverted chromosomal segment in susceptible compared to resistant tomato

The gene Mi-1 confers effective resistance in tomato (Lycopersicon esculentum) against root-knot nematodes and some isolates of potato aphid. This locus was introgressed from L. peruvianum into the corresponding region on chromosome 6 in tomato. In nematode-resistant tomato, Mi-1 and six homologs are grouped into two clusters separated by 300 kb. Analysis of BAC clones revealed that the Mi-1 locus from susceptible tomato carried the same number and distribution of Mi-1 homologs, as did the resistant locus. Molecular markers flanking the resistant and susceptible loci were in the same relative orientation, but markers between the two clusters were in an inverse orientation. The simplest explanation for these observations is that there is an inversion between the two clusters of homologs when comparing the Mi-1 loci from L. esculentum and L. peruvianum. Such an inversion may explain previous observations of severe recombination suppression in the region. Two Mi-1 homologs identified from the BAC library derived from susceptible tomato are not linked to the chromosome 6 locus, but map to chromosome 5 in regions known to contain resistance gene loci in other solanaceous species.Communicated by J.S. Heslop-Harrison     

Genetic diversity and morphological differentiation in Liatris helleri (Asteraceae), a threatened plant species

Liatris helleri (Asteraceae) is an insect-pollinated herbaceous perennial endemic to several high-elevation sites in the Blue Ridge Mountains of North Carolina. Allozymes were used to describe the genetic diversity and population structure in nine populations of this rare, federally listed species. Differences in leaf morphology were also examined for greenhouse-grown plants representing several populations. The proportion of the total genetic diversity found among populations, as indicated by the allozyme data, was 16%. Higher levels of population differentiation were found for differences in leaf shape; population of origin accounted for 37% of the variation in maximum leaf width, while families within populations accounted for 7%. In contrast to many endemic species, L. helleri maintains fairly high levels of genetic diversity. For the species, the percent polymorphic loci was 87.5, the average number of alleles at variable loci was 3.00 and the gene diversity was 0.276. Mean population values were percent polymorphic loci =58.4, mean number of alleles per polymorphic locus =2.59 and gene diversity =0.219. The estimated gene flow was low (Nm=0.85–1.32) and a relatively high correlation (r=0.55; p<0.005) was found between linear geographic and genetic distance. This suggests that the populations are partially isolated by distance, despite the limited range (<60 km) of the species. We recommend that population distinetiveness be maintained in restoration efforts.     

Assessment of gene copy number in the homosporous fernsCeratopteris thalictroides andC. richardii (Parkeriaceae) by restriction fragment length polymorphisms

Homosporous ferns are generally considered polyploid due to high chromosome numbers, but genetically diploid since the expression of isozymes is generally controlled by a single locus. Gene silencing over evolutionary time is one means by which this apparent contradiction can be explained. A prediction of this hypothesis is that silenced gene sequences still reside in the genomes of homosporous ferns. We examined the genomes ofCeratopteris richardii andC. thalictroides for sequences which are similar to expressed gene sequences. Genomic DNA blots hybridized withC. richardii cDNA clones showed that the majority of these clones detected multiple fragments, suggesting that most gene-like sequences are duplicated inCeratopteris. Hybridization signal intensity often varied between fragments of the same size between accessions, sometimes dramatically, which indicates that not all sequences are equivalent, and may represent the products of silenced genes. Observed reciprocal differences in intensity could be due to reciprocally silenced genes. In addition, an unusual segregation pattern for one locus followed by one probe may indicate homeologous chromosome pairing and segregation.     

Distinguishing allozymes and isozymes of phosphoglucoisomerases by electrophoretic comparisons of pollen and somatic tissues

The different electrophoretic patterns of dimeric phosphoglucoisomerases extracted from haploid pollen and diploid somatic tissues of plants may be used to distinguish allozymes and isozymes. The analysis depends on the presence of two alleles at each locus in somatic tissues but only one or the other allele in pollen grains. Consequently, in heterozygotes, heterodimeric allozymes can be identified because they are formed in stems and leaves but not in pollen. The procedure is described in enzymes extracted from the diploid annual plant Clarkia dudleyana, which possesses three gene loci for PGI subunits. Comparison of the electrophoretic patterns of stem and pollen extracts makes it possible in many cases to identify allelic state without breeding tests. The technique also is likely to be useful in the interpretation of zymograms of other multimeric enzymes coded by more than one gene locus.     

Organization of phenylalanine ammonia lyase (<Emphasis Type="Italic">PAL</Emphasis>), acidic <Emphasis Type="Italic">PR-5</Emphasis> and osmotin-like (<Emphasis Type="Italic">OSM</Emphasis>) defence-response gene families in the potato genome

Defence-response (DR) genes are candidates for the genetic functions underlying quantitative resistance to plant pathogens. The organization of three DR gene families encoding phenylalanine ammonia lyase (PAL), acidic PR-(pathogenesis-related) protein 5, and basic PR-5, or osmotin-like (OSM), proteins was studied in the potato genome. A bacterial artificial chromosome (BAC) library containing ~50,000 clones was constructed from high-molecular weight genomic DNA of the diploid potato clone PD59, a hybrid between Solanum tuberosum and S. phureja. BAC clones carrying one or more copies of the DR genes were identified and characterized by Southern hybridization, sequence analysis and genetic mapping. PAL, acidic PR-5 and OSM (basic PR-5) genes were all organized into gene families of varying complexity. The PAL gene family consisted of at least 16 members, several of which were physically linked. Four acidic PR-5 homologous were localized to a 45-kb segment on potato chromosome XII. One of these, PR-5/319, codes for the acidic thaumatin-like protein C found in intercellular fluids of potato. Nine OSM genes were organized at two loci: eight form a 90-kb cluster on chromosome VIII, and a single gene was found on chromosome XI. The topology of a phylogenetic tree based on PR-5 and OSM protein sequences from Solanaceae suggests a mode of evolution for these gene families. The results will form the basis for further studies on the potential role of these defence-related loci in quantitative resistance to pathogens.     

Molecular analysis of single copy and repetitive genes on chromosome 2 in intergeneric tomato somatic hybrid plants

Restriction fragment polymorphisms were used to identify and quantify the nuclear contributions from each parent to somatic hybrid plants between tomato (Lycopersicon esculentum Mill.) cv. Sub-Arctic Maxi and Solanum lycopersicoides Dun. Three single-copy clones, 2–13, 2–17, and 3–288, and a clone for the 45s ribosomal RNA, pHA2, all mapped to chromosome 2 of tomato, were used in analysis of 47 somatic hybrids. The amount of hybridizing probe for each parental band was quantified by densitometry of the autoradiograph film. Analyses with the three single-copy clones indicated that there were more than two S. lycopersicoides copies in most somatic hybrid plants. For at least one somatic hybrid there was a loss of one tomato copy. No evidence was found for more than two copies donated from tomato or loss of a copy from S. lycopersicoides. Most of the observed variation in copy number of the single-copy clones was consistent with chromosomal changes occurring in the suspension cells from which S. lycopersicoides parental protoplasts were derived.The number of copies of rDNA derived from each parent varied independently of the number of copies of single-copy clones from each parent. Changes in the copy number of rDNA occurred in both tomato and S. lycopersicoides genomes.     

A molecular and cytogenetic survey of major repeated DNA sequences in tomato (Lycopersicon esculentum)

Summary The major families of repeated DNA sequences in the genome of tomato (Lycopersicon esculentum) were isolated from a sheared DNA library. One thousand clones, representing one million base pairs, or 0.15% of the genome, were surveyed for repeated DNA sequences by hybridization to total nuclear DNA. Four major repeat classes were identified and characterized with respect to copy number, chromosomal localization by in situ hybridization, and evolution in the family Solanaceae. The most highly repeated sequence, with approximately 77000 copies, consists of a 162 bp tandemly repeated satellite DNA. This repeat is clustered at or near the telomeres of most chromosomes and also at the centromeres and interstitial sites of a few chromosomes. Another family of tandemly repeated sequences consists of the genes coding for the 45 S ribosomal RNA. The 9.1 kb repeating unit in L. esculentum was estimated to be present in approximately 2300 copies. The single locus, previously mapped using restriction fragment length polymorphisms, was shown by in situ hybridization as a very intense signal at the end of chromosome 2. The third family of repeated sequences was interspersed throughout nearly all chromosomes with an average of 133 kb between elements. The total copy number in the genome is approximately 4200. The fourth class consists of another interspersed repeat showing clustering at or near the centromeres in several chromosomes. This repeat had a copy number of approximately 2100. Sequences homologous to the 45 S ribosomal DNA showed cross-hybridization to DNA from all solanaceous species examined including potato, Datura, Petunia, tobacco and pepper. In contrast, with the exception of one class of interspersed repeats which is present in potato, all other repetitive sequences appear to be limited to the crossing-range of tomato. These results, along with those from a companion paper (Zamir and Tanksley 1988), indicate that tomato possesses few highly repetitive DNA sequences and those that do exist are evolving at a rate higher than most other genomic sequences.     

Molecular and genetic characterization of elite transgenic rice plants produced by electric-discharge particle acceleration

The recovery of transgenic rice plants expressing a number of exogenous genes was reported previously. Using immature embryo explants as the target tissue, plasmids containing both selectable and screenable marker genes were introduced into elite rice varieties via electric-discharge particle acceleration. Co-integration, copy number, expression, and inheritance of these genes were analyzed. A 100% co-integration frequency was confirmed by Southern-blot analyses of R0 plants. The majority of transgenic plants contained between one and ten copies of exogenous DNA and molecular and genetic analyses of progeny indicated that all copies in almost all R0 plants were inherited as a single dominant hemizygous locus. Co-expression of unselected genes ranged from 30–66% for gus/hmr constructs, depending on the promotor used, and up to 90% for bar/hmr constructs. The integrative structures of two unlinked transgenic loci of a rare R0 plant were analyzed in detail by Southern-blot analysis of its progeny.     

Patterns of genome duplication within the Brassica napus genome.

The progenitor diploid genomes (A and C) of the amphidiploid Brassica napus are extensively duplicated with 73% of genomic clones detecting two or more duplicate sequences within each of the diploid genomes. This comprehensive duplication of loci is to be expected in a species that has evolved through a polyploid ancestor. The majority of the duplicate loci within each of the diploid genomes were found in distinct linkage groups as collinear blocks of linked loci, some of which had undergone a variety of rearrangements subsequent to duplication, including inversions and translocations. A number of identical rearrangements were observed in the two diploid genomes, suggesting they had occurred before the divergence of the two species. A number of linkage groups displayed an organization consistent with centric fusion and (or) fission, suggesting this mechanism may have played a role in the evolution of Brassica genomes. For almost every genetically mapped locus detected in the A genome a homologous locus was found in the C genome; the collinear arrangement of these homologous markers allowed the primary regions of homoeology between the two genomes to be identified. At least 16 gross chromosomal rearrangements differentiated the two diploid genomes during their divergence from a common ancestor.     

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.     

Structure of the multigene family of MAL loci in Saccharomyces

Summary Multigene families are a ubiquitous feature of eukaryotes; however, their presence in Saccharomyces is more limited. The MAL multigene family is comprised of five unlined loci, MAL1, MAL2, MAL3, MAL4 and MAL6, any one of which is sufficient for yeast to metabolize maltose. A cloned MAL6 locus was used as a probe to facilitate the cloning of the other four functional loci as well as two partially active alleles of MAL1. Each locus could be characterized as a cluster of three genes, MALR (regulatory), MALT (maltose transport or permease) and MALS (structural or maltase), encoded by a total of about 7 kb of DNA; however, homologous sequences at each locus extend beyond the coding regions. Our results indicate that there is extensive homology among the MAL loci, especially within their maltase genes. The greatest sequence diversity occurs in their regulatory gene regions. Southern cross analyses of the cloned MAL loci indicate a single duplication of the MAL6R-homologous sequences upstream of the MAL6R gene as well as an extensive duplication of more than 10 kb at the MAL3 locus. The large repeat at the MAL3 locus results in the presence of four copies of MAL3R-homologous sequences and two copies of MAL3T-homologous sequences at that locus. Two naturally occurring inactive alleles of MAL1 show a deletion or divergence of their MALR sequences. The significance of these repeats in the evolution of the MAL multigene family is discussed.     

Aspartate aminotransferase isozymes in the genusCapsella (Brassicaceae): Subcellular location,gene duplication,and polymorphism

The subcellular location of aspartate aminotransferase isozymes (EC 2.6.1.1) in the genusCapsella(Brassicaceae) was studied. The diploid speciesC. grandiflora andC. rubella have three AAT isozymes, including one located in the plastids. Each locus is duplicated in the tetraploidCapsella bursa-pastoris. Variation at the plastid-coding locus exceeded that at the other loci.C. bursa-pastoris had some unique alleles not detected in the diploid species. Segregation in open-pollinated families revealed thatCapsella grandiflora was outcrossing, whereasC. rubella was highly inbred, with most populations homozygous or uniform at all three loci. Inheritance in the tetraploid colonizerC. bursa-pastoris is disomic. This species was also predominantly selfing with outcrossing rates between 2% and 10%.Financial support by the German Research Foundation DFG is gratefully acknowledged.     

Isolation of homozygous transgenicBrassica napus lines carrying a seed-specific chimeric 2S albumin gene and determination of linkage relationships

UsingAgrobacterium tumefaciens-mediated gene transfer, 14 T0Brassica napus plants carrying one of three chimeric 2S albumin seed protein genes were obtained after co-transformation with theneo gene. Plants were made homozygous using either cell haploid culture or, for single-copy plants, traditional crossing methods. Sixty-five percent of kanamycin-resistant plants contained at least one copy of the seed protein gene, and multiple copies were usually at a single locus. In the majority of cases, at least one copy of theneo gene was linked to an introduced 2S albumin gene, demonstrating that co-transformation is not a reliable way to obtain lines without the marker gene linked to the gene of interest. In 3 T0 plants sequences derived from beyond the left border of the vector were integrated in the plant genome, in two cases partially rearranged, confirming that T-DNA insertion is not always precise. Procedures for efficiently determining this are described. This work highlights the extra steps needed to prepare transgenic lines for field studies and potential further breeding.     

Do potatoes and tomatoes have a single evolutionary history, and what proportion of the genome supports this history?

Background  

Phylogenies reconstructed with only one or a few independently inherited loci may be unresolved or incongruent due to taxon and gene sampling, horizontal gene transfer, or differential selection and lineage sorting at individual loci. In an effort to remedy this situation, we examined the utility of conserved orthologous set (COSII) nuclear loci to elucidate the phylogenetic relationships among 29 diploid Solanum species in the sister clades that include tomato and potato, and in Datura inoxia as a far outgroup. We screened 40 COSII markers with intron content over 60% that are mapped in different chromosomes; selected a subset of 19 by the presence of single band amplification of size mostly between 600 and 1200 bp; sequenced these 19 COSII markers, and performed phylogenetic analyses with individual and concatenated datasets. The present study attempts to provide a fully resolved phylogeny among the main clades in potato and tomato that can help to identify the appropriate markers for future studies using additional species.     

Gene silencing in potato: allelic differences and effect of ploidy

Silencing of genes is mostly studied in diploid, homozygous, self-fertile and sexually propagated species. However, conclusions drawn for these species are not always applicable to crops like potato, which is an autotetraploid, highly heterozygous, vegetatively propagated species. Factors influencing the level of silencing in potato are discussed, with emphasis on inhibition of the granule-bound starch synthase I (GBSSI) gene. Type of construct, number of integrated T-DNA copies, structural arrangement of the T-DNA locus, integration site, target tissue and genetic background are important factors for all plant species. Ploidy level and multiple allelism are factors deserving special attention when the efficiency of silencing of endogenous genes is studied in polyploid, heterozygous species such as potato.