Recent amplification of triose phosphate isomerase related sequences in lettuce
'
ILANPARAN, RICHARD V. KESSELI,~ LOREWESTPHAL,AND RICHARD W.
MICHELMORE~
Department of Vegetable Crops, University of California, Davis, CA 95616, U.S.A . Corresponding Editor: R. Kemble Received December 10, 1991
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Accepted March 27, 1992 PARAN,I., KESSELI,R. V., WESTPHAL,L., and MICHELMORE, R. W. 1992. Recent amplification of triose phosphate isomerase related sequences in lettuce. Genome, 35: 627-635. A random cDNA clone was identified as distinguishing near-isogenic lines for downy mildew resistance in lettuce. The clone detected multiple restriction fragments in genomic Southern blots of lettuce. Restriction fragment length polymorphisms (RFLPs) detected by this clone mapped to separate clusters of resistance genes; therefore, these sequences were studied in a greater detail. Sequence analysis indicated that the cDNA encoded the glycolytic enzyme triose phosphate isomerase (TPI). The lettuce clone shares 85% sequence similarity at the amino acid level with TPI from maize. TPIrelated sequences were mapped in lettuce using three crosses. Ten loci were distributed in six linkage groups. Possible mechanisms of amplification and dispersion were investigated. Retrotransposition was excluded, since intron five is retained in all TPI-related genomic sequences. Large scale chromosomal rearrangements were not involved, as RFLP markers flanking TPI loci were not duplicated. A high level of genomic variability was detected by the TPI clone; 37 different restriction fragments were detected in Southern hybridizations to 64 populations of lettuce including 47 cultivars of Lactuca sativa and five wild species. Species distantly related t o L. sativa had few TPI loci, indicating that their amplification and dispersion were recent and had occurred after the emergence of the L. serriola complex. Key words: triose phosphate isomerase, gene duplication, lettuce. PARAN,I., KESSELI,R. V., WESTPHAL,L., et MICHELMORE, R. W. 1992. Recent amplification of triose phosphate isomerase related sequences in lettuce. Genome, 35 : 627-635. Un clone casualis6 d'ADNc a CtC identifik, permettant de distinguer des lignCes presque isogkniques pour la rCsistance au mildiou de la laitue Le clone a dCtectC des fragments de restriction multiples dans des buvardages de type Southern de laitue. Les longueurs polymorphes de fragments de restriction (PLFR) dCtectCes par ce clone ont CtC cartographikes pour sCparer les groupes de genes de rksistance, puis ces skquences ont CtC CtudiCes plus en dCtail. L'analyse des sCquences a indiquC que I'ADNc encodait l'enzyme glycolytique triose phosphate isomCrase (TPI). La similarit6 des skquences de la laitue au niveau des acides aminCs avec celles de la TPI du mais a CtC de 85%. Trois croisements de laitue ont CtC utilisCs pour cartographier les skquences relikes a la TPI. Dix locus Ctaient distribuCs dans six groupes de linkage. Les mCcanismes de dispersion et d'amplification possibles ont CtC investiguks. La retransposition a CtC exclue, puisque l'intron 5 Ctait retenu chez toutes les TPI des skquences gknomiques reliCes. Les rkarrangements chromosomiques a grande Cchelle n'ont pas CtC impliquCs, vu que les marqueurs de PLFR de part et d'autre des locus des TPI n'ont pas CtC dupliquks. Un niveau ClevC de variabilitk gCnomique a CtC dCcelC par le clone de TPI; 37 fragments de restriction diffkrents ont CtC dCtectCs par des hybridations de type Southern chez 64 populations de laitue, incluant 47 cultivars de Lactuca sativa et cinq especes indigenes. Les especes relikes de f a ~ o nCloignCe au L. sativa avaient peu de locus TPI, ce qui indique que leur dispersion et leur amplification ont CtC rCcentes et qu'elles sont survenues apres 1'Cmergence du complexe L. serriola. Mots clbs : triose phosphate isomkrase, duplication des gknes, laitue. [Traduit par la rkdaction]
Introduction We are establishing detailed genetic maps in the genus Lactuca (Landry et al. 1987a; Kesseli et al. 1991) with particular emphasis on disease resistance genes. The maps are composed of restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), isozyme, and morphological markers as well as disease resistance genes. In the course of saturating the regions containing resistance genes with markers, we isolated a random cDNA clone that distinguished near-isogenic lines for downy mildew resistance (Paran et al. 1991). This clone detected RFLP loci that mapped to different clusters of resistance genes. Some resistance genes in plants may be members of 'present address: Department of and Hebrew University of Jerusalem, Rehovot, Israel, 76100. address: Department of Biology, University of Massachusetts, Boston, MA 02125-3393, U.S.A. 'present address: Institut fur Angewandte Genetik, Universitat Hannover, 3000 Hannover 2 1, Germany. ' ~ u t h o r to whom all correspondence should be addressed. Pr~ntedIn Canada / lrnpr~rneau Canada
multigene families that have diverged to have different specificities (Michelmore et al. 1987; Pryor 1987). Therefore, this cDNA sequence was characterized and potential mechanisms of amplification and dispersal were studied further. The clone showed a high level of sequence similarity to the glycolytic enzyme' triose phosphate isomerase (TPI). TPI catalyses the reversible isomerization of glyceraldehyde3-phosphate and dihydroxyacetone phosphate in the glycolytic and gluconeogenic pathways (Noltmann 1972). Mammals have multiple isozymes of TPI that are posttranslational modifications of a protein derived from a single active nuclear gene (Brown et al. 1985; Bulfield et al. 1987). Southern analysis with probes for TPI revealed multiple bands in human (Maquat et al. 1985). Several copies of the TPI-related sequences in human have been shown to be processed pseudogenes (Brown et 1985), iSe., nonfunctional genes that are similar in structure to mRNA and thus presumably derived from mRNA by reverse transcription. the human TPI probe was used in genetic mapping of mouse; the TPI probe detected nine loci that were distributed on seven chromosomes (Siracusa et al. 1991).
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Multiple copies of TPI-related sequences are also present in the maize genome, the only plant species from which the TPI gene has been isolated to date (Marchionni and Gilbert 1986); however, the basis of the amplification in maize is unknown. All higher plants have at least two isozymes of TPI, one located in the plastid and one located in the cytosol (Pichersky and Gottlieb 1984). The two proteins are each encoded by separate nuclear genes that assorted independently in Clarkia spp. and Stephanomeria diegensis (Pichersky and Gottlieb 1983; Gallez and Gottlieb 1982). Duplications of the genes that encode the cytosolic or the plastid isozymes have been reported in maize and in Clarkia (Wendel et al. 1989; Pichersky and Gottlieb 1983). In both species, the duplicated genes are not linked. The two TPI isozymes from lettuce have been purified and are composed of two subunits of molecular mass 27 000 Da (Pichersky and Gottlieb 1984). Isozyme analysis indicated that only two active forms of TPI exist in lettuce ( 0 . Ochoa and R.W. Michelmore, unpublished; Pichersky and Gottlieb 1984). In this paper we describe multiple TPI-related sequences scattered throughout the genome of lettuce. No evidence was found for the existence of intronless TPI genes or for duplication of markers flanking TPI loci. Therefore, the existence of processed pseudogenes of TPI or large chromosomal rearrangements as causes for genomic amplification were ruled out. Large numbers of RFLPs were detected within Lactuca spp. using a probe for TPI. The number of TPI-related sequences varied greatly between related Lactuca species, indicating recent amplification of these sequences.
Materials and methods Stocks and crosses Three F2 populations were used for genetic analysis: 1, 66 plants of an intraspecific cross of Lactuca sativa cv. Calmar x cv. Kordaat (Landry et al. 1987a); 2, 158 plants of an interspecific cross between L. sativa x L. saligna cv. Vanguard 75 x UC82US1; 3, 57 plants of an intraspecific cross of L. saligna, UC82US1 x PI261653. The 47 cultivars of Lactuca sativa representing different types of the cultivated lettuce and 18 accessions of wild Lactuca species that were used in this study are described elsewhere (Kesseli et al. 1991). Each cultivar or accession was a bulk of at least 20 plants so that the major alleles characteristic of each line would be detected. DNA isolation, restriction digests, Southern blotting, probe labeling, and hybridization Plant DNA was isolated by the CTAB method as described by Bernatzky and Tanksley (1986), omitting the last step of CsCl ethidium bromide centrifugation. Digested genomic DNA (7 pg/ lane) was fractionated in a 1% agarose gel and blotted onto either Zetaprobe (Bio-Rad) or Hybond N + (Amersham) membranes. DNA probes, cloned in either pARC7 (Landry et al. 1987b) or pBS + / - (Stratagene), were isolated using the Geneclean kit (Bio-101 Inc.). The probes were labelled by the random primer method (Feinberg and Vogelstein 1983) using the Amersham kit and hybridized to Southern blots using standard conditions (Landry et al. 1987b). A genomic library of lettuce in Charon 4A (B.S. Landry and R.W. Michelmore, unpublished) was plated on nitrocellulose filters and was screened using methods described in Sambrook et al. (1989). DNA extraction of positive lambda clones was done according to the plate lysate protocol in Sambrook et al. (1989). Restriction mapping was done by single and double restriction enzymes digestions followed by 1% gel electrophoresis. Other procedures were as described previously (Landry et al. 1987b).
VOL. 35,
1992
Isozyme analysis Electrophoresis procedures were as described by Kesseli and Michelmore (1986). Electrode buffer, gel buffer, and staining were prepared as described in Wendel et al. (1989). All tissue extractions were performed on seedlings of F3 families. For each family a minimum of five F3 plants were bulked together to determine the progenitor F2 genotype. Clone isolation and sequencing pCL1795, a clone from the cDNA library of lettuce (Paran et al. 1991), was subcloned into the pBS + / - vector (Stratagene) and deletions were made using exonuclease I11 and S l nuclease (Heinikoff 1984). Double strand sequencing from overlapping dones was performed according to the dideoxy termination method (Sanger et al. 1977) using the Sequenase kit (USB). Homology search was made using the Wisconsin Genetics Computer Group (GCG) program (Devereux et al. 1984). PCR amplification Polymerase chain reactions (PCRs) were performed using lambda genomic clones or genomic DNA (50 ng) as templates in a Perkin Elmer Cetus DNA thermal cycler. Two 20-mer oligonucleotide primers were synthesized (Operon Technologies Inc., Alameda, Calif.) using- seauences within exons five and six in the clone * pCL1795. The nucleotide sequences of the two primers were TTTGTTGGAGACAAGGTTGC and CAATAGCCCAAACTGGCTCA. Reaction conditions were 1 min at 94OC, 1 min at 55"C, and 1 min at 72OC for 30 cycles followed by 1 cycle of 5 min at 72°C. PCR products were analyzed by 2% agarose gel electrophoresis and visualized with UV after ethidium bromide staining. PCR products were gel purified, treated with T4 polynucleotide kinase, and cloned into a SmaI site of pBS + / - for sequencing. Linkage analysis Segregation of RFLP and isozyme loci was analyzed using the computer program MAPMAKER (Lander et al. 1987). Linkage with a LOD score of at least three was considered significant.
Results T o obtain markers that are linked to genes for resistance to downy mildew (Dm), a cDNA library of lettuce was screened for probes that detected polymorphisms between near-isogenic lines that differed for particular resistance genes (Dm3 and D m l ) (Paran et al. 1991). Among other clones, pCL1795 was identified as detecting polymorphism between the near-isogenic lines (Paran et al. 1991). This probe detected multiple bands and RFLPs in Southern blots. Segregation analysis indicated that loci detected by pCL1795 were linked to two different linkage groups of Dm genes. We therefore investigated pCL1795 further and determined its sequence. We determined the genomic distribution of restriction fragments detected by pCL1795. We then investigated possible mechanisms of amplification and the evolution of this gene family. Sequence of pCL1795 The lettuce cDNA clone is 785 bp long. An open reading frame of 585 bp is followed by a 200-bp 3' nontranslated region. pCL1795 shares sequence similarity with TPI from all organisms for which TPI sequences are available. The greatest similarity is with TPI from maize, 85% at the amino acid level (Fig. I), the only other plant sequence available. Similarity to chicken is 54% and to human and rabbit 5 1%. From the sizes of the expected protein in lettuce (Pichersky and Goettlieb 1984), and the maize gene, the first 176 bp of the coding region are missing from pCL1795. Our let-
PARAN ET AL.
ATCCAAGTTGCAGCTCAAAATTGTTGGGTTAAGAAGGGTGGTGCATTCACCGGTGAGGTTAGTGCTGAGATG
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720 785
FIG. 1. Sequence similarity of triose phosphate isomerase from lettuce and maize. The nucleotide sequence of the lettuce cDNA, pCL1795, is shown with the deduced amino acid sequence below (le). This is similar to the amino acid sequence of TPI from maize (ma) except for the first 59 amino acids missing from the lettuce clone. Colons between letters indicate identical amino acids. A single dot indicates a missing amino acid. The positions of the two oligonucleotide primers used for PCR amplification of intron five are underlined.
tuce cDNA library was screened with pCL1795; however, no larger clones were obtained. Linkage analysis of TPI-related sequences Some of the loci of the multigene family detected by pCL1795 were mapped using three crosses with which we are establishing detailed genetic maps. In the Calmar x Kordaat cross, 15 fragments were present in a HindIII digest of which five were polymorphic. In UC82US1 x Vanguard 75, four of nine fragments were polymorphic in a EcoRI
digest. In UC82US1 x PI261653, five of eight fragments were polymorphic in a HindIII digest. Polymorphic restriction fragments were initially scored as present or absent because it was unknown which restriction fragments were allelic. For one locus, CL1795H14,both alleles were identified and therefore they could be rescored as codominant. It is likely that some loci were monomorphic in these analyses and therefore their position could not be determined. A total of 10 loci could be analyzed in the three crosses. These loci were distributed in six of the different major
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GENOME, VOL. 35, 1992
FIG. 2. Distribution of TPI-related sequences in the lettuce genome. The genetic positions of RFLPs detected by pCL1795 are shown within the 1 1 major linkage groups currently identified in lettuce (n = 9, Kesseli et al. 1990). These TPI-related sequences were mapped in at least one of three crosses: A, UC82US1 x P1261653; , Calmar x Kordaat; *, UC82USl x Vanguard 75.
linkage groups so far identified (Fig. 2). CL1795H48was unlinked to any of the 62 mapped markers in UC82US1 x PI261653. Only one locus, ~ ~ 1 7 9 5 , ~segregated ,, in two crosses; all the other loci segregated in only one of the three crosses. The parents of the three crosses were also analyzed for TPI isozymes. Only the locus for the plastid isozyme of TPI (the faster migrating isozyme; Pichersky and Gottlieb 1984) was polymorphic and segregated in the interspecific and intraspecific crosses involving L. saligna. This locus cosegregated with CL 1795E28and was designated Tpil . Conservation of intron five in TPZ We investigated whether amplification of TPI-related sequences may have resulted from retrotransposition to unlinked sites by determining if the lettuce TPI gene family contained sequences lacking introns. Retrotransposition involves RNA-dependent DNA replication and random integration of the duplicated sequences into the genome. The consequences of this type of amplification are that the duplicated genes lack introns and untranscribed regulatory sequences. Since the positions of five of the seven introns of TPI are identical in maize and chicken (Marchionni and Gilbert 1986), it was probable that these intron boundaries were also conserved in lettuce. A pair of 20-mer oligonucleotide primers was synthesized (underlined in Fig. 1) that corresponded to sequences in exons five and six in pCL 1795 that flank intron five and are completely conserved between maize and lettuce. Intron five and flanking sequences were amplified by PCR using genomic clones of TPI as templates. A genomic library of lettuce in lambdaphage, Charon 4A (approximately 5 x 10' clones) was screened with pCL1795. Thirty-six positive lambda clones were isolated and analyzed with restriction endonucleases and hybridization to pCL 1795. Four distinct clones were identified based on their restriction maps and pattern of hybridization. PCR amplification resulted in fragments of the same size, 370 bp, from all four genomic clones. This was larger than the 190-bp product obtained when pCL1795 was used as a template (Fig. 3a), indicating the presence of an approximately 180-bp intron in all the genomic clones. The amplification products were subcloned and sequenced to verify the positions of the intron boundaries. Intron five in all four genomic clones had an identical, 73% AT rich, 174-bp sequence (Fig. 4a) in the
1 2 3 4 5 6
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FIG. 3. Products of PCR amplification using primers from exons five and six of triose phosphate isomerase. Lane 1 , 123 bp molecular weight ladder; lane 2, amplification product using the cDNA, pCL1795 as template. (A) Lanes 3-6: amplification products using four different genomic lambda clones (Fig. 3) as templates. (B) Lane 3: amplification product using a genomic lambda clone of TPI as template; lanes 4-6: amplification products using genomic DNA from L. sativa, L. saligna, and L. serriola, respectively.
predicted position. The 100- and 33-bp sequences of the two flanking exons were identical to the cDNA sequence of pCL 1795. Intron five was also amplified by PCR using total genomic DNA from three species: L. sativa, L. serriola, and L. saligna, as templates. Two amplification products were observed: a 370-bp band that was identical in size to the amplified product from the genomic lambda clones of TPI
PARAN ET AL.
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FIG. 4. Sequences of the two products from genomic PCR amplification using primers in exons five and six of triose phosphate isomerase. (A) Nucleotide sequences of intron five: a, 370-bp amplification product; b , 300-bp amplification product. A vertical line indicates identical bases. The positions of DraI and HinfI sites are marked with a A . (B) Nucleotide sequences and deduced amino acid sequences of exons five and six from the two amplification products: a, 370-bp product; b, 300-bp product. The position of intron five is marked with a A. Vertical lines indicate identical amino acids. A dot indicates conserved amino acids.
and a second smaller band of approximately 300 bp (Fig. 3b). There was no 190-bp product characteristic of a sequence lacking in intron. No polymorphism was observed between the three species examined. The two PCR products from L. sativa were excised from the gel and cloned. Three independent clones were sequenced for each product. The sequence of the 370-bp genomic PCR product included intron five and the two flanking exons and was identical to the sequence of the PCR product from the lambda clones. The 300-bp PCR product contained exon sequences and a smaller intron of 116 bp (Fig. 4a). The two introns shared 47% sequence similarity (Fig. 4a) and were in the same position in the open reading frame. In addition, there were also differences between the flanking exons in the two amplification products at 19 positions, resulting in predicted changes of five amino acids (Fig. 4b). A 145-bp DraI-HinfI internal fragment of the amplified 174-bp intron (Fig. 4a) was subcloned and used as a probe for hybridization to Southern blots of lettuce genomic DNA. The hybridization pattern with this intron probe was identical to the pattern detected when the cDNA pCL1795 was used as a probe (Fig. 5). These results provided further
evidence that no intronless TPI-related sequences were detectable in the lettuce genome. Variability in TPZ-related sequences DNA from 47 cultivars of L. sativa representing different types of lettuce and from five related wild species was digested with two restriction enzymes, EcoRI and HindIII. Southern blots were probed with pCL1795 (Fig. 6). The combined data from both the HindIII and EcoRI digestions showed that 42 of the 47 lines of L. sativa had unique genotypes detected by pCL1795. The HindIII digestion alone provided unique genotypes for 38 of the lines (Table I). All the wild accessions also had unique genotypes. Lactuca serriola was the most variable species of the sampled populations; 29 different polymorphic restriction fragments were detected in L. serriola, while 16 were detected in L. sativa, 10 in L. saligna and 3 in L. virosa. The total number of different restriction fragments in all the sampled populations was 37. There was, however, a difference between the mean number of restriction fragments within each species (Table I). More than 10 restriction fragments per population were detected in L. sativa and L. serriola in the HindIII
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GENOME, VOL. 35, 1992
FIG. 5. Hybridization of cDNA and intron five of TPI to Southern blots of lettuce genomic DNA. DNA of two F, individuals from a cross between cv. Saffier and PIVT1309 was digested with Hind111 and probed with the cDNA contained in pCL1795 (panel 1). The blot was strip washed and then reprobed with the 145-bp HinfI-DraI internal fragment within the 174-bp intron five of TPI (panel 2). digestion, while only 6 were detected in L. saligna and one each in L. virosa, L. perennis, and L. indica. Discussion Sequences related to TPI are dispersed through the genome of lettuce. Linkage analysis identified loci in six major linkage groups. Tight physical clustering was not observed as no more than one TPI-related sequence was detected on each lambda clone. It is not known how many of these sequences are actively transcribed. All of the genomic sequences apparently contained intron five. Isozyme analysis indicated that activity of the plastid form segregated as a single locus and .therefore is encoded by a single gene or a cluster of genes. Isozyme polymorphism was not detected for the cytosolic form and therefore it could not be analyzed genetically. Unlinked duplications have been reported for several genes across a wide variety of plant taxa. Isozyrne analysis detected unlinked duplications of genes encoding phosphoglucose isomerase (PGI), 6-phosphogluconate dehydrogenase (6-PGD), and phosphoglucose mutase (PGM) in Clarkia (Gottlieb 1982; Odrzykoski and Gottlieb 1984; Soltis et al. 1987). Genes that encoded for photosynthetic proteins such as the small subunit of ribulose biphosphate carboxylase (RBCS) and chlorophyll a/b binding protein (CAB) have been duplicated to independent loci in tomato (Tanksley and Pichersky 1988). In lettuce, at least seven unlinked duplications were detected by RFLP mapping of 200 random cDNA clones (Kesseli et al. 1990; R.V. Kesseli and R.W. Michelmore, unpublished). Similar analysis revealed a higher percentage of unlinked duplicated RFLP loci in tomato and maize, 20 and 29070, respectively (Pichersky 1990). There is little experimental evidence, however, regarding the mechanisms by which these duplications have occurred.
There are several mechanisms by which duplication of unlinked genes may occur (Pichersky 1990). One mechanism involves chromosomal rearrangements, such as translocations between nonhomologous chromosomes. This mechanism is likely to produce large duplicated segments. The genetic map of lettuce (Kesseli et al. 1990) has no indication for duplications of large linkage blocks including the TPI-related genes. Therefore, chromosomal rearrangements are unlikely to explain the extensive amplification of the TPI-related loci. At least two types of transposition can result in amplification of genomic sequences to unlinked positions. Retrotransposition is caused by a RNA-mediated DNA duplication (Maeda and Smithies 1986) in which double-stranded DNA is synthesized from RNA and is randomly integrated into the genome. In mammals, interspersed DNA sequences such as the Alu family and processed pseudogenes are postulated to arise by this mechanism (Rogers 1985). Although processed pseudogenes were detected in the TPI multigene family in humans, our data for TPI in lettuce provide no evidence for retrotransposition since all the TPI genes detected contained at least intron five and PCR failed to amplify sequences lacking intron five from genomic DNA. A second form of transposition, nonreplicative transposition, can also result in amplification of sequences. Several families of transposons have been demonstrated throughout the genomes of diverse eukaryotes such as Drosophila and maize (Finnegan 1985). Sequences may become trapped within transposons and amplified along with the element. The variation in copy number of TPIrelated sequences within and between different Lactuca species without duplication of flanking markers suggests that some form of transposition may be occurring. However, it is difficult to demonstrate the involvement of transposition in amplification of sequences unrelated to a transposon. At a minimum, it would require extensive segregation analysis, cross-hybridization studies, and sequencing of several genomic clones to determine the extent of duplicated sequences flanking the TPI genes and to search for sequence motifs that are characteristic of transposons. Two distinct TPI-related sequences were detected by PCR analysis of genomic DNA using primers in exons five and six. The two amplified products differed in the size and sequence of intron five as well as in the exon sequences. The flanking exon sequences shared 86% sequence similarity over 133 bases; the sequence differences would result in five differences at the amino acid level and were all at positions that were unconserved between lettuce and corn. The two products amplified by PCR could have been derived from several types of diverged sequences. Plant species have two genes for TPI, one encoding the cytosolic enzyme the other encoding the plastid enzyme. Therefore, the two amplification products may represent the two isozymes. In the two species where genes encoding both the cytosolic and the plastid isozymes have been sequenced, they shared only partial sequence similarity to each other: 58% between isozymes of phosphoglucoisomerase in Clarkia spp. (Gottlieb 1988) and 45% between isozymes of glucose-3phosphate dehydrogenase in tobacco (Shin et al. 1986). Alternatively, both amplification products may have been derived from sequences that diverged after amplification of a gene encoding a single isozyme. Additional TPI-related sequences may exist in the lettuce genome. However, the identical hybridization patterns between the cDNA clone and
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FIG. 6. Hybridization of pCL1795 to Southern blots of genomic DNA of lettuce cultivars and wild species of Lactuca digested with HindIII. Lanes 1-1 1: Butterhead cultivars Bourguignonne Grosse Blonde d'Hiver, Hilde, Kordaat, Lednicky, Mariska, May King, Mildura, Passion Blonde, Sabine, Saffier, Santa-Anna, respectively. Lanes 12-1 9: L. serriola LSE 18, LSE57/ 15, PIVT1309, PI 171669, PI25 1245, PI255665, PI281877, PI190906, respectively. Lane 20: L. virosa UC83UK1. Lane 21: Crisphead cultivar Calmar. TABLE1. Variability of TPI-related sequences in HindIII digestion of genomic DNA of Lactuca species
Species
No. of unique genotypes/ species
Total no. of bands/ species
38 8 4 2 1 1
21 29 11 2 1 1
No. of polymorphic bands within species
No. of bands/ population Min
Max
Mean
-
L. L. L. L. L. L.
sativa (n = 47)* serriola (n = 8) saligna (n = 4) virosa (n = 3) perennis (n = 1) indica (n = 1)
sequences from the larger intron indicated that additional TPI-related sequences are either not numerous or are greatly diverged. The pCL1795 clone detected frequent RFLPs. All except two of the fragments detected in a HindIII digest of L. sativa were also detected in L. serriola; this reflects the involvement of L. serriola in the origin of L. sativa (Ryder and Whitaker 1976; Kesseli et al. 1991). The majority (89%) of lettuce cultivars and all the wild species accessions could be distinguished from each other by hybridization of Southern blots with pCL1795. Of the approximately 2000 random cDNAs that we have screened during the development of the genetic map of lettuce, pCL1795 detected the most
polymorphic banding pattern and may therefore be useful for fingerprinting lettuce cultivars. The six species of Lactuca included in this study differed from each other in the average number of restriction fragments detected in Southern blots hybridized with pCL1795. Lactuca serriola, L. sativa, L. saligna, and L. virosa are all part of a complex of species with varying degrees of sexual intercompatibility (Ryder and Whitaker 1976). Lactuca perennis and L. indica are more distantly related species (Kesseli et al. 1991). All these species are 2n = 18. While multiple HindIII fragments were always detected in Southern blots of L. sativa, L. serriola, and L. saligna (mean number = 14, 11, and 6, respectively), only one fragment
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was detected in accessions of L. virosa, L. perennis, and L. indica. This may indicate that pCL1795 detected only one of the two genes encoding TPI. When the hybridization stringency was reduced to washing conditions of 55"C/1 x SSC, only two addition restriction fragments were detected in L. perennis and L. virosa. Therefore, the large variation for the number of TPI-related sequences can only be partially attributed to loss of homology to pCL1795. The amplification of TPI-related sequences therefore occurred recently after the emergence of the L. serriola complex. The lack of sequence variation in intron five from four independent genomic clones also indicates that the amplification event was recent. The variation in the number of TPI-related sequences within L. sativa, L. serriola, and L. saligna may indicate that amplification is still continuing. Studies on isozyme duplications in Clarkia and their phylogenetic implications demonstrated that most gene duplications were restricted to the Clarkia genus or to sections of Clarkia, but not to other related genera (Pichersky and Gottlieb 1983; Gottlieb 1988), indicating that these duplications also had a relatively recent origin.
Acknowledgments We thank Oswaldo Ochoa for technical assistance. This research was supported by a grant from the U.S. Department of Agriculture (88-CRCR-37262-3522). Bernatzky, R., and Tanksley, S.D. 1986. Genetics of actin related sequences in tomato. Theor. Appl. Genet. 72: 314-321. Brown, J.R., Daar, I.O., Krug, J.R., and Maquat, L.E. 1985. Characterization of the functional gene and several processed pseudogenes in the human triosephosphate isomerase gene family. Mol. Cell. Biol. 5: 1694-1706. Bulfield, G., Ball, S.T., and Peters, J. 1987. An allele at the triosephosphate isomerase, TPI-I locus on chromosome 6 recovered from feral mice. Genet. Res. 50: 239-243. Devereux, J., Haeberli, P., and Smithies, 0. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12: 387-395. Feinberg, A.P., and Vogelstein, B. 1983. A technique for radiolabelling DNA restriction fragments to high specific activity. Anal. Biochem. 132: 6-13. Finnegan, D.J. 1985. Transposable elements in eukaryotes. Int. Rev.. Cytol. 93: 281-326. Gallez, G.P., and Gottlieb, L.D. 1982. Genetic evidence for the hybrid origin of the diploid plant Stephanomeria diegensis. Evolution, 36: 1158-1 167. Gottlieb, L.D. 1982. Conservation and duplication of isozymes in plants. Science (Washington, D.C.), 216: 373-380. Gottlieb, L.D. 1988. Towards molecular genetics in Clarkia: gene duplications and molecular characterization of PGI genes. Ann. Mo. Bot. Gard. 75: 1169-1179. Heinikoff, S. 1984. Unidirectional digestion with exonuclease I11 creates targeted breakpoints for DNA sequencing. Gene, 28: 351-359. Kesseli, R.V., and Michelmore, R. W. 1986. Genetic variation and phylogenies detected from isozyme markers in species of Lactuca. Hered. 77: 324-331. Kesseli, R.V., Paran, I., and Michelmore, R.W. 1990. Lactuca sativa. In Genetic maps. 5th ed. Edited by S.J. O'Brien. Cold Spring Harbor Press, N.Y. pp. 6.100-6.102. Kesseli, R. V., Paran, I., and Michelmore, R. W. 1991. The variation at RFLP loci in Lactuca spp. and origin of cultivated lettuce (L. sativa). Genome, 34: 430-436.
Lander, E.S., Green, P., Abrahamson, J., et al. 1987. MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics, 1: 174- 181. Landry, B.S., Kesseli, R., Farrara, B., and Michelmore, R.W. 1987a. A genetic map of lettuce (Lactuca sativa L.). with restriction fragment length polymorphism, isozyme, disease resistance and morphological markers. Genetics, 116: 33 1-337. Landry, B.S., Kesseli, R., Leung, H., and Michelmore, R.W. 19876. Comparison of restriction endonucleases and sources of probes for their efficiency in detecting restriction fragment length polymorphisms in lettuce (Lactuca sativa L.). Theor. Appl. Genet. 74: 646-653. Maeda, N., and Smithies, 0. 1986. The evolution of multigene families: human haptoglobin genes. Ann. Rev. Genet. 20: 81-108. Maquat, L.E., Chilcote, R., and Ryan, P.M. 1985. Human triosephosphate isomerase cDNA and protein structure. J. Biol. Chem. 260: 3748-3753. Marchionni, M., and Gilbert, W. 1986. The triosephosphate isomerase gene from maize: introns antedate the plant-animal divergence. Cell, 46: 133- 141. Michelmore, R.W., Hulbert, S.H., Landry B.S., and Leung, H. 1987. Towards a molecular understanding of lettuce downy mildew. In Genetics and plant pathogenesis. Edited by P.R. Day and G. J . Jelis. Blackwell Scientific Publications, Oxford. pp. 221-231. Noltmann, E.A. 1972. Aldose ketose isomerases. Enzymes, 1: 271-353. Odrzykoski, I. J., and Gottlieb, L.D. 1984. Duplications of genes encoding 6-phosphogluconate dehydrogenase in Clarkia (Onagraceae) and their phylogenetic implications. Syst. Bot. 9: 479-489. Paran, I., Kesseli, R.V., and Michelmore, R. W. 1991. Identification of restriction fragment length polymorphism and random amplified polymorphic DNA markers linked to downy mildew resistance genes in lettuce using near-isogenic lines. Genome, 34: 1021-1027. Pichersky, E. 1990. Nomad DNA-A model for movement and duplication of DNA sequences in plant genomes. Plant Mol. Biol. 15: 437-448. Pichersky, E., and Gottlieb, L.D. 1983. Evidence for duplication of the structural genes coding plastid and cytosolic isozymes of triose phosphate isomerase in diploid species of Clarkia. Genetics 105: 421-436. Pichersky, E., and Gottlieb, L.D. 1984. Plant triose phosphate isomerase isozymes. Plant Physiol. 74: 340-347. Pryor, T. 1987. The origin and structure of fungal disease resistance genes in plants. Trends Genet. 3: 157-161. Rogers, J.H. 1985. The origin and evolution of retroposons. Int. Rev. Cytol. 93: 187-279. Ryder, E. J., and Whitaker, T. W. 1976. Lettuce (Lactuca sativa). In Evolution of crop plants. Edited by N.W. Simmonds. Longman Scientific and Technical, Edinburgh. pp. 39-41. Sambrook, J., Maniatis, T., and Fritsch, F.F. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York. Sanger, F., Nicklen, S., and Coulsen, A.R. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74: 5463-5467. Shin, C.H., Lazar, G., and Goodman, H.M. 1986. Evidence in favor of the symbiotic origin of chloroplasts: primary structure and evolution of tobacco glyceraldehyde-2-phosphate dehydrogenases. Cell, 47: 73-80. Siracusa, L.D., Jenkins, N.A., and Copeland, N.G. 1991. Identification and application of repetitive probes for gene mapping in the mouse. Genetics, 127: 169-179. Soltis, P.A., Soltis, D.E., and Gottlieb, L.D. 1987. Phosphoglucose
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mutase gene duplications in Clarkia (Onagraceae) and their phylogenetic implications. Evolution, 41: 667-67 1. Tanksley, S.D., and Pichersky, E. 1988. Organization and evolution of sequences in the plant nuclear genome. I n Plant evolutionary biology. Edited by L.D. Gottlieb and S. Jain. Chapman and Hill, Inc., London. pp. 55-83.
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Wendel, J.F., Stuber, C.W., Goodman, M.M., and Beckett, J.B. 1989. Duplicated plastid and triplicated cytosolic isozymes of triosephosphate isomerase in maize (Zea mays L.). J. Hered. 80: 21 8-228.
'
ILANPARAN, RICHARD V. KESSELI,~ LOREWESTPHAL,AND RICHARD W.
MICHELMORE~
Department of Vegetable Crops, University of California, Davis, CA 95616, U.S.A . Corresponding Editor: R. Kemble Received December 10, 1991
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Accepted March 27, 1992 PARAN,I., KESSELI,R. V., WESTPHAL,L., and MICHELMORE, R. W. 1992. Recent amplification of triose phosphate isomerase related sequences in lettuce. Genome, 35: 627-635. A random cDNA clone was identified as distinguishing near-isogenic lines for downy mildew resistance in lettuce. The clone detected multiple restriction fragments in genomic Southern blots of lettuce. Restriction fragment length polymorphisms (RFLPs) detected by this clone mapped to separate clusters of resistance genes; therefore, these sequences were studied in a greater detail. Sequence analysis indicated that the cDNA encoded the glycolytic enzyme triose phosphate isomerase (TPI). The lettuce clone shares 85% sequence similarity at the amino acid level with TPI from maize. TPIrelated sequences were mapped in lettuce using three crosses. Ten loci were distributed in six linkage groups. Possible mechanisms of amplification and dispersion were investigated. Retrotransposition was excluded, since intron five is retained in all TPI-related genomic sequences. Large scale chromosomal rearrangements were not involved, as RFLP markers flanking TPI loci were not duplicated. A high level of genomic variability was detected by the TPI clone; 37 different restriction fragments were detected in Southern hybridizations to 64 populations of lettuce including 47 cultivars of Lactuca sativa and five wild species. Species distantly related t o L. sativa had few TPI loci, indicating that their amplification and dispersion were recent and had occurred after the emergence of the L. serriola complex. Key words: triose phosphate isomerase, gene duplication, lettuce. PARAN,I., KESSELI,R. V., WESTPHAL,L., et MICHELMORE, R. W. 1992. Recent amplification of triose phosphate isomerase related sequences in lettuce. Genome, 35 : 627-635. Un clone casualis6 d'ADNc a CtC identifik, permettant de distinguer des lignCes presque isogkniques pour la rCsistance au mildiou de la laitue Le clone a dCtectC des fragments de restriction multiples dans des buvardages de type Southern de laitue. Les longueurs polymorphes de fragments de restriction (PLFR) dCtectCes par ce clone ont CtC cartographikes pour sCparer les groupes de genes de rksistance, puis ces skquences ont CtC CtudiCes plus en dCtail. L'analyse des sCquences a indiquC que I'ADNc encodait l'enzyme glycolytique triose phosphate isomCrase (TPI). La similarit6 des skquences de la laitue au niveau des acides aminCs avec celles de la TPI du mais a CtC de 85%. Trois croisements de laitue ont CtC utilisCs pour cartographier les skquences relikes a la TPI. Dix locus Ctaient distribuCs dans six groupes de linkage. Les mCcanismes de dispersion et d'amplification possibles ont CtC investiguks. La retransposition a CtC exclue, puisque l'intron 5 Ctait retenu chez toutes les TPI des skquences gknomiques reliCes. Les rkarrangements chromosomiques a grande Cchelle n'ont pas CtC impliquCs, vu que les marqueurs de PLFR de part et d'autre des locus des TPI n'ont pas CtC dupliquks. Un niveau ClevC de variabilitk gCnomique a CtC dCcelC par le clone de TPI; 37 fragments de restriction diffkrents ont CtC dCtectCs par des hybridations de type Southern chez 64 populations de laitue, incluant 47 cultivars de Lactuca sativa et cinq especes indigenes. Les especes relikes de f a ~ o nCloignCe au L. sativa avaient peu de locus TPI, ce qui indique que leur dispersion et leur amplification ont CtC rCcentes et qu'elles sont survenues apres 1'Cmergence du complexe L. serriola. Mots clbs : triose phosphate isomkrase, duplication des gknes, laitue. [Traduit par la rkdaction]
Introduction We are establishing detailed genetic maps in the genus Lactuca (Landry et al. 1987a; Kesseli et al. 1991) with particular emphasis on disease resistance genes. The maps are composed of restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), isozyme, and morphological markers as well as disease resistance genes. In the course of saturating the regions containing resistance genes with markers, we isolated a random cDNA clone that distinguished near-isogenic lines for downy mildew resistance (Paran et al. 1991). This clone detected RFLP loci that mapped to different clusters of resistance genes. Some resistance genes in plants may be members of 'present address: Department of and Hebrew University of Jerusalem, Rehovot, Israel, 76100. address: Department of Biology, University of Massachusetts, Boston, MA 02125-3393, U.S.A. 'present address: Institut fur Angewandte Genetik, Universitat Hannover, 3000 Hannover 2 1, Germany. ' ~ u t h o r to whom all correspondence should be addressed. Pr~ntedIn Canada / lrnpr~rneau Canada
multigene families that have diverged to have different specificities (Michelmore et al. 1987; Pryor 1987). Therefore, this cDNA sequence was characterized and potential mechanisms of amplification and dispersal were studied further. The clone showed a high level of sequence similarity to the glycolytic enzyme' triose phosphate isomerase (TPI). TPI catalyses the reversible isomerization of glyceraldehyde3-phosphate and dihydroxyacetone phosphate in the glycolytic and gluconeogenic pathways (Noltmann 1972). Mammals have multiple isozymes of TPI that are posttranslational modifications of a protein derived from a single active nuclear gene (Brown et al. 1985; Bulfield et al. 1987). Southern analysis with probes for TPI revealed multiple bands in human (Maquat et al. 1985). Several copies of the TPI-related sequences in human have been shown to be processed pseudogenes (Brown et 1985), iSe., nonfunctional genes that are similar in structure to mRNA and thus presumably derived from mRNA by reverse transcription. the human TPI probe was used in genetic mapping of mouse; the TPI probe detected nine loci that were distributed on seven chromosomes (Siracusa et al. 1991).
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Multiple copies of TPI-related sequences are also present in the maize genome, the only plant species from which the TPI gene has been isolated to date (Marchionni and Gilbert 1986); however, the basis of the amplification in maize is unknown. All higher plants have at least two isozymes of TPI, one located in the plastid and one located in the cytosol (Pichersky and Gottlieb 1984). The two proteins are each encoded by separate nuclear genes that assorted independently in Clarkia spp. and Stephanomeria diegensis (Pichersky and Gottlieb 1983; Gallez and Gottlieb 1982). Duplications of the genes that encode the cytosolic or the plastid isozymes have been reported in maize and in Clarkia (Wendel et al. 1989; Pichersky and Gottlieb 1983). In both species, the duplicated genes are not linked. The two TPI isozymes from lettuce have been purified and are composed of two subunits of molecular mass 27 000 Da (Pichersky and Gottlieb 1984). Isozyme analysis indicated that only two active forms of TPI exist in lettuce ( 0 . Ochoa and R.W. Michelmore, unpublished; Pichersky and Gottlieb 1984). In this paper we describe multiple TPI-related sequences scattered throughout the genome of lettuce. No evidence was found for the existence of intronless TPI genes or for duplication of markers flanking TPI loci. Therefore, the existence of processed pseudogenes of TPI or large chromosomal rearrangements as causes for genomic amplification were ruled out. Large numbers of RFLPs were detected within Lactuca spp. using a probe for TPI. The number of TPI-related sequences varied greatly between related Lactuca species, indicating recent amplification of these sequences.
Materials and methods Stocks and crosses Three F2 populations were used for genetic analysis: 1, 66 plants of an intraspecific cross of Lactuca sativa cv. Calmar x cv. Kordaat (Landry et al. 1987a); 2, 158 plants of an interspecific cross between L. sativa x L. saligna cv. Vanguard 75 x UC82US1; 3, 57 plants of an intraspecific cross of L. saligna, UC82US1 x PI261653. The 47 cultivars of Lactuca sativa representing different types of the cultivated lettuce and 18 accessions of wild Lactuca species that were used in this study are described elsewhere (Kesseli et al. 1991). Each cultivar or accession was a bulk of at least 20 plants so that the major alleles characteristic of each line would be detected. DNA isolation, restriction digests, Southern blotting, probe labeling, and hybridization Plant DNA was isolated by the CTAB method as described by Bernatzky and Tanksley (1986), omitting the last step of CsCl ethidium bromide centrifugation. Digested genomic DNA (7 pg/ lane) was fractionated in a 1% agarose gel and blotted onto either Zetaprobe (Bio-Rad) or Hybond N + (Amersham) membranes. DNA probes, cloned in either pARC7 (Landry et al. 1987b) or pBS + / - (Stratagene), were isolated using the Geneclean kit (Bio-101 Inc.). The probes were labelled by the random primer method (Feinberg and Vogelstein 1983) using the Amersham kit and hybridized to Southern blots using standard conditions (Landry et al. 1987b). A genomic library of lettuce in Charon 4A (B.S. Landry and R.W. Michelmore, unpublished) was plated on nitrocellulose filters and was screened using methods described in Sambrook et al. (1989). DNA extraction of positive lambda clones was done according to the plate lysate protocol in Sambrook et al. (1989). Restriction mapping was done by single and double restriction enzymes digestions followed by 1% gel electrophoresis. Other procedures were as described previously (Landry et al. 1987b).
VOL. 35,
1992
Isozyme analysis Electrophoresis procedures were as described by Kesseli and Michelmore (1986). Electrode buffer, gel buffer, and staining were prepared as described in Wendel et al. (1989). All tissue extractions were performed on seedlings of F3 families. For each family a minimum of five F3 plants were bulked together to determine the progenitor F2 genotype. Clone isolation and sequencing pCL1795, a clone from the cDNA library of lettuce (Paran et al. 1991), was subcloned into the pBS + / - vector (Stratagene) and deletions were made using exonuclease I11 and S l nuclease (Heinikoff 1984). Double strand sequencing from overlapping dones was performed according to the dideoxy termination method (Sanger et al. 1977) using the Sequenase kit (USB). Homology search was made using the Wisconsin Genetics Computer Group (GCG) program (Devereux et al. 1984). PCR amplification Polymerase chain reactions (PCRs) were performed using lambda genomic clones or genomic DNA (50 ng) as templates in a Perkin Elmer Cetus DNA thermal cycler. Two 20-mer oligonucleotide primers were synthesized (Operon Technologies Inc., Alameda, Calif.) using- seauences within exons five and six in the clone * pCL1795. The nucleotide sequences of the two primers were TTTGTTGGAGACAAGGTTGC and CAATAGCCCAAACTGGCTCA. Reaction conditions were 1 min at 94OC, 1 min at 55"C, and 1 min at 72OC for 30 cycles followed by 1 cycle of 5 min at 72°C. PCR products were analyzed by 2% agarose gel electrophoresis and visualized with UV after ethidium bromide staining. PCR products were gel purified, treated with T4 polynucleotide kinase, and cloned into a SmaI site of pBS + / - for sequencing. Linkage analysis Segregation of RFLP and isozyme loci was analyzed using the computer program MAPMAKER (Lander et al. 1987). Linkage with a LOD score of at least three was considered significant.
Results T o obtain markers that are linked to genes for resistance to downy mildew (Dm), a cDNA library of lettuce was screened for probes that detected polymorphisms between near-isogenic lines that differed for particular resistance genes (Dm3 and D m l ) (Paran et al. 1991). Among other clones, pCL1795 was identified as detecting polymorphism between the near-isogenic lines (Paran et al. 1991). This probe detected multiple bands and RFLPs in Southern blots. Segregation analysis indicated that loci detected by pCL1795 were linked to two different linkage groups of Dm genes. We therefore investigated pCL1795 further and determined its sequence. We determined the genomic distribution of restriction fragments detected by pCL1795. We then investigated possible mechanisms of amplification and the evolution of this gene family. Sequence of pCL1795 The lettuce cDNA clone is 785 bp long. An open reading frame of 585 bp is followed by a 200-bp 3' nontranslated region. pCL1795 shares sequence similarity with TPI from all organisms for which TPI sequences are available. The greatest similarity is with TPI from maize, 85% at the amino acid level (Fig. I), the only other plant sequence available. Similarity to chicken is 54% and to human and rabbit 5 1%. From the sizes of the expected protein in lettuce (Pichersky and Goettlieb 1984), and the maize gene, the first 176 bp of the coding region are missing from pCL1795. Our let-
PARAN ET AL.
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FIG. 1. Sequence similarity of triose phosphate isomerase from lettuce and maize. The nucleotide sequence of the lettuce cDNA, pCL1795, is shown with the deduced amino acid sequence below (le). This is similar to the amino acid sequence of TPI from maize (ma) except for the first 59 amino acids missing from the lettuce clone. Colons between letters indicate identical amino acids. A single dot indicates a missing amino acid. The positions of the two oligonucleotide primers used for PCR amplification of intron five are underlined.
tuce cDNA library was screened with pCL1795; however, no larger clones were obtained. Linkage analysis of TPI-related sequences Some of the loci of the multigene family detected by pCL1795 were mapped using three crosses with which we are establishing detailed genetic maps. In the Calmar x Kordaat cross, 15 fragments were present in a HindIII digest of which five were polymorphic. In UC82US1 x Vanguard 75, four of nine fragments were polymorphic in a EcoRI
digest. In UC82US1 x PI261653, five of eight fragments were polymorphic in a HindIII digest. Polymorphic restriction fragments were initially scored as present or absent because it was unknown which restriction fragments were allelic. For one locus, CL1795H14,both alleles were identified and therefore they could be rescored as codominant. It is likely that some loci were monomorphic in these analyses and therefore their position could not be determined. A total of 10 loci could be analyzed in the three crosses. These loci were distributed in six of the different major
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GENOME, VOL. 35, 1992
FIG. 2. Distribution of TPI-related sequences in the lettuce genome. The genetic positions of RFLPs detected by pCL1795 are shown within the 1 1 major linkage groups currently identified in lettuce (n = 9, Kesseli et al. 1990). These TPI-related sequences were mapped in at least one of three crosses: A, UC82US1 x P1261653; , Calmar x Kordaat; *, UC82USl x Vanguard 75.
linkage groups so far identified (Fig. 2). CL1795H48was unlinked to any of the 62 mapped markers in UC82US1 x PI261653. Only one locus, ~ ~ 1 7 9 5 , ~segregated ,, in two crosses; all the other loci segregated in only one of the three crosses. The parents of the three crosses were also analyzed for TPI isozymes. Only the locus for the plastid isozyme of TPI (the faster migrating isozyme; Pichersky and Gottlieb 1984) was polymorphic and segregated in the interspecific and intraspecific crosses involving L. saligna. This locus cosegregated with CL 1795E28and was designated Tpil . Conservation of intron five in TPZ We investigated whether amplification of TPI-related sequences may have resulted from retrotransposition to unlinked sites by determining if the lettuce TPI gene family contained sequences lacking introns. Retrotransposition involves RNA-dependent DNA replication and random integration of the duplicated sequences into the genome. The consequences of this type of amplification are that the duplicated genes lack introns and untranscribed regulatory sequences. Since the positions of five of the seven introns of TPI are identical in maize and chicken (Marchionni and Gilbert 1986), it was probable that these intron boundaries were also conserved in lettuce. A pair of 20-mer oligonucleotide primers was synthesized (underlined in Fig. 1) that corresponded to sequences in exons five and six in pCL 1795 that flank intron five and are completely conserved between maize and lettuce. Intron five and flanking sequences were amplified by PCR using genomic clones of TPI as templates. A genomic library of lettuce in lambdaphage, Charon 4A (approximately 5 x 10' clones) was screened with pCL1795. Thirty-six positive lambda clones were isolated and analyzed with restriction endonucleases and hybridization to pCL 1795. Four distinct clones were identified based on their restriction maps and pattern of hybridization. PCR amplification resulted in fragments of the same size, 370 bp, from all four genomic clones. This was larger than the 190-bp product obtained when pCL1795 was used as a template (Fig. 3a), indicating the presence of an approximately 180-bp intron in all the genomic clones. The amplification products were subcloned and sequenced to verify the positions of the intron boundaries. Intron five in all four genomic clones had an identical, 73% AT rich, 174-bp sequence (Fig. 4a) in the
1 2 3 4 5 6
1 2 3 4 5 6
FIG. 3. Products of PCR amplification using primers from exons five and six of triose phosphate isomerase. Lane 1 , 123 bp molecular weight ladder; lane 2, amplification product using the cDNA, pCL1795 as template. (A) Lanes 3-6: amplification products using four different genomic lambda clones (Fig. 3) as templates. (B) Lane 3: amplification product using a genomic lambda clone of TPI as template; lanes 4-6: amplification products using genomic DNA from L. sativa, L. saligna, and L. serriola, respectively.
predicted position. The 100- and 33-bp sequences of the two flanking exons were identical to the cDNA sequence of pCL 1795. Intron five was also amplified by PCR using total genomic DNA from three species: L. sativa, L. serriola, and L. saligna, as templates. Two amplification products were observed: a 370-bp band that was identical in size to the amplified product from the genomic lambda clones of TPI
PARAN ET AL.
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I IIIII II I l l I II II I I I I I I I I I I I I I ACACCAAAATCATAACTTTTGAGTGCAAGTTACTTATTATATCMGATMCAAGATTTTGAT
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FIG. 4. Sequences of the two products from genomic PCR amplification using primers in exons five and six of triose phosphate isomerase. (A) Nucleotide sequences of intron five: a, 370-bp amplification product; b , 300-bp amplification product. A vertical line indicates identical bases. The positions of DraI and HinfI sites are marked with a A . (B) Nucleotide sequences and deduced amino acid sequences of exons five and six from the two amplification products: a, 370-bp product; b, 300-bp product. The position of intron five is marked with a A. Vertical lines indicate identical amino acids. A dot indicates conserved amino acids.
and a second smaller band of approximately 300 bp (Fig. 3b). There was no 190-bp product characteristic of a sequence lacking in intron. No polymorphism was observed between the three species examined. The two PCR products from L. sativa were excised from the gel and cloned. Three independent clones were sequenced for each product. The sequence of the 370-bp genomic PCR product included intron five and the two flanking exons and was identical to the sequence of the PCR product from the lambda clones. The 300-bp PCR product contained exon sequences and a smaller intron of 116 bp (Fig. 4a). The two introns shared 47% sequence similarity (Fig. 4a) and were in the same position in the open reading frame. In addition, there were also differences between the flanking exons in the two amplification products at 19 positions, resulting in predicted changes of five amino acids (Fig. 4b). A 145-bp DraI-HinfI internal fragment of the amplified 174-bp intron (Fig. 4a) was subcloned and used as a probe for hybridization to Southern blots of lettuce genomic DNA. The hybridization pattern with this intron probe was identical to the pattern detected when the cDNA pCL1795 was used as a probe (Fig. 5). These results provided further
evidence that no intronless TPI-related sequences were detectable in the lettuce genome. Variability in TPZ-related sequences DNA from 47 cultivars of L. sativa representing different types of lettuce and from five related wild species was digested with two restriction enzymes, EcoRI and HindIII. Southern blots were probed with pCL1795 (Fig. 6). The combined data from both the HindIII and EcoRI digestions showed that 42 of the 47 lines of L. sativa had unique genotypes detected by pCL1795. The HindIII digestion alone provided unique genotypes for 38 of the lines (Table I). All the wild accessions also had unique genotypes. Lactuca serriola was the most variable species of the sampled populations; 29 different polymorphic restriction fragments were detected in L. serriola, while 16 were detected in L. sativa, 10 in L. saligna and 3 in L. virosa. The total number of different restriction fragments in all the sampled populations was 37. There was, however, a difference between the mean number of restriction fragments within each species (Table I). More than 10 restriction fragments per population were detected in L. sativa and L. serriola in the HindIII
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GENOME, VOL. 35, 1992
FIG. 5. Hybridization of cDNA and intron five of TPI to Southern blots of lettuce genomic DNA. DNA of two F, individuals from a cross between cv. Saffier and PIVT1309 was digested with Hind111 and probed with the cDNA contained in pCL1795 (panel 1). The blot was strip washed and then reprobed with the 145-bp HinfI-DraI internal fragment within the 174-bp intron five of TPI (panel 2). digestion, while only 6 were detected in L. saligna and one each in L. virosa, L. perennis, and L. indica. Discussion Sequences related to TPI are dispersed through the genome of lettuce. Linkage analysis identified loci in six major linkage groups. Tight physical clustering was not observed as no more than one TPI-related sequence was detected on each lambda clone. It is not known how many of these sequences are actively transcribed. All of the genomic sequences apparently contained intron five. Isozyme analysis indicated that activity of the plastid form segregated as a single locus and .therefore is encoded by a single gene or a cluster of genes. Isozyme polymorphism was not detected for the cytosolic form and therefore it could not be analyzed genetically. Unlinked duplications have been reported for several genes across a wide variety of plant taxa. Isozyrne analysis detected unlinked duplications of genes encoding phosphoglucose isomerase (PGI), 6-phosphogluconate dehydrogenase (6-PGD), and phosphoglucose mutase (PGM) in Clarkia (Gottlieb 1982; Odrzykoski and Gottlieb 1984; Soltis et al. 1987). Genes that encoded for photosynthetic proteins such as the small subunit of ribulose biphosphate carboxylase (RBCS) and chlorophyll a/b binding protein (CAB) have been duplicated to independent loci in tomato (Tanksley and Pichersky 1988). In lettuce, at least seven unlinked duplications were detected by RFLP mapping of 200 random cDNA clones (Kesseli et al. 1990; R.V. Kesseli and R.W. Michelmore, unpublished). Similar analysis revealed a higher percentage of unlinked duplicated RFLP loci in tomato and maize, 20 and 29070, respectively (Pichersky 1990). There is little experimental evidence, however, regarding the mechanisms by which these duplications have occurred.
There are several mechanisms by which duplication of unlinked genes may occur (Pichersky 1990). One mechanism involves chromosomal rearrangements, such as translocations between nonhomologous chromosomes. This mechanism is likely to produce large duplicated segments. The genetic map of lettuce (Kesseli et al. 1990) has no indication for duplications of large linkage blocks including the TPI-related genes. Therefore, chromosomal rearrangements are unlikely to explain the extensive amplification of the TPI-related loci. At least two types of transposition can result in amplification of genomic sequences to unlinked positions. Retrotransposition is caused by a RNA-mediated DNA duplication (Maeda and Smithies 1986) in which double-stranded DNA is synthesized from RNA and is randomly integrated into the genome. In mammals, interspersed DNA sequences such as the Alu family and processed pseudogenes are postulated to arise by this mechanism (Rogers 1985). Although processed pseudogenes were detected in the TPI multigene family in humans, our data for TPI in lettuce provide no evidence for retrotransposition since all the TPI genes detected contained at least intron five and PCR failed to amplify sequences lacking intron five from genomic DNA. A second form of transposition, nonreplicative transposition, can also result in amplification of sequences. Several families of transposons have been demonstrated throughout the genomes of diverse eukaryotes such as Drosophila and maize (Finnegan 1985). Sequences may become trapped within transposons and amplified along with the element. The variation in copy number of TPIrelated sequences within and between different Lactuca species without duplication of flanking markers suggests that some form of transposition may be occurring. However, it is difficult to demonstrate the involvement of transposition in amplification of sequences unrelated to a transposon. At a minimum, it would require extensive segregation analysis, cross-hybridization studies, and sequencing of several genomic clones to determine the extent of duplicated sequences flanking the TPI genes and to search for sequence motifs that are characteristic of transposons. Two distinct TPI-related sequences were detected by PCR analysis of genomic DNA using primers in exons five and six. The two amplified products differed in the size and sequence of intron five as well as in the exon sequences. The flanking exon sequences shared 86% sequence similarity over 133 bases; the sequence differences would result in five differences at the amino acid level and were all at positions that were unconserved between lettuce and corn. The two products amplified by PCR could have been derived from several types of diverged sequences. Plant species have two genes for TPI, one encoding the cytosolic enzyme the other encoding the plastid enzyme. Therefore, the two amplification products may represent the two isozymes. In the two species where genes encoding both the cytosolic and the plastid isozymes have been sequenced, they shared only partial sequence similarity to each other: 58% between isozymes of phosphoglucoisomerase in Clarkia spp. (Gottlieb 1988) and 45% between isozymes of glucose-3phosphate dehydrogenase in tobacco (Shin et al. 1986). Alternatively, both amplification products may have been derived from sequences that diverged after amplification of a gene encoding a single isozyme. Additional TPI-related sequences may exist in the lettuce genome. However, the identical hybridization patterns between the cDNA clone and
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FIG. 6. Hybridization of pCL1795 to Southern blots of genomic DNA of lettuce cultivars and wild species of Lactuca digested with HindIII. Lanes 1-1 1: Butterhead cultivars Bourguignonne Grosse Blonde d'Hiver, Hilde, Kordaat, Lednicky, Mariska, May King, Mildura, Passion Blonde, Sabine, Saffier, Santa-Anna, respectively. Lanes 12-1 9: L. serriola LSE 18, LSE57/ 15, PIVT1309, PI 171669, PI25 1245, PI255665, PI281877, PI190906, respectively. Lane 20: L. virosa UC83UK1. Lane 21: Crisphead cultivar Calmar. TABLE1. Variability of TPI-related sequences in HindIII digestion of genomic DNA of Lactuca species
Species
No. of unique genotypes/ species
Total no. of bands/ species
38 8 4 2 1 1
21 29 11 2 1 1
No. of polymorphic bands within species
No. of bands/ population Min
Max
Mean
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L. L. L. L. L. L.
sativa (n = 47)* serriola (n = 8) saligna (n = 4) virosa (n = 3) perennis (n = 1) indica (n = 1)
sequences from the larger intron indicated that additional TPI-related sequences are either not numerous or are greatly diverged. The pCL1795 clone detected frequent RFLPs. All except two of the fragments detected in a HindIII digest of L. sativa were also detected in L. serriola; this reflects the involvement of L. serriola in the origin of L. sativa (Ryder and Whitaker 1976; Kesseli et al. 1991). The majority (89%) of lettuce cultivars and all the wild species accessions could be distinguished from each other by hybridization of Southern blots with pCL1795. Of the approximately 2000 random cDNAs that we have screened during the development of the genetic map of lettuce, pCL1795 detected the most
polymorphic banding pattern and may therefore be useful for fingerprinting lettuce cultivars. The six species of Lactuca included in this study differed from each other in the average number of restriction fragments detected in Southern blots hybridized with pCL1795. Lactuca serriola, L. sativa, L. saligna, and L. virosa are all part of a complex of species with varying degrees of sexual intercompatibility (Ryder and Whitaker 1976). Lactuca perennis and L. indica are more distantly related species (Kesseli et al. 1991). All these species are 2n = 18. While multiple HindIII fragments were always detected in Southern blots of L. sativa, L. serriola, and L. saligna (mean number = 14, 11, and 6, respectively), only one fragment
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was detected in accessions of L. virosa, L. perennis, and L. indica. This may indicate that pCL1795 detected only one of the two genes encoding TPI. When the hybridization stringency was reduced to washing conditions of 55"C/1 x SSC, only two addition restriction fragments were detected in L. perennis and L. virosa. Therefore, the large variation for the number of TPI-related sequences can only be partially attributed to loss of homology to pCL1795. The amplification of TPI-related sequences therefore occurred recently after the emergence of the L. serriola complex. The lack of sequence variation in intron five from four independent genomic clones also indicates that the amplification event was recent. The variation in the number of TPI-related sequences within L. sativa, L. serriola, and L. saligna may indicate that amplification is still continuing. Studies on isozyme duplications in Clarkia and their phylogenetic implications demonstrated that most gene duplications were restricted to the Clarkia genus or to sections of Clarkia, but not to other related genera (Pichersky and Gottlieb 1983; Gottlieb 1988), indicating that these duplications also had a relatively recent origin.
Acknowledgments We thank Oswaldo Ochoa for technical assistance. This research was supported by a grant from the U.S. Department of Agriculture (88-CRCR-37262-3522). Bernatzky, R., and Tanksley, S.D. 1986. Genetics of actin related sequences in tomato. Theor. Appl. Genet. 72: 314-321. Brown, J.R., Daar, I.O., Krug, J.R., and Maquat, L.E. 1985. Characterization of the functional gene and several processed pseudogenes in the human triosephosphate isomerase gene family. Mol. Cell. Biol. 5: 1694-1706. Bulfield, G., Ball, S.T., and Peters, J. 1987. An allele at the triosephosphate isomerase, TPI-I locus on chromosome 6 recovered from feral mice. Genet. Res. 50: 239-243. Devereux, J., Haeberli, P., and Smithies, 0. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12: 387-395. Feinberg, A.P., and Vogelstein, B. 1983. A technique for radiolabelling DNA restriction fragments to high specific activity. Anal. Biochem. 132: 6-13. Finnegan, D.J. 1985. Transposable elements in eukaryotes. Int. Rev.. Cytol. 93: 281-326. Gallez, G.P., and Gottlieb, L.D. 1982. Genetic evidence for the hybrid origin of the diploid plant Stephanomeria diegensis. Evolution, 36: 1158-1 167. Gottlieb, L.D. 1982. Conservation and duplication of isozymes in plants. Science (Washington, D.C.), 216: 373-380. Gottlieb, L.D. 1988. Towards molecular genetics in Clarkia: gene duplications and molecular characterization of PGI genes. Ann. Mo. Bot. Gard. 75: 1169-1179. Heinikoff, S. 1984. Unidirectional digestion with exonuclease I11 creates targeted breakpoints for DNA sequencing. Gene, 28: 351-359. Kesseli, R.V., and Michelmore, R. W. 1986. Genetic variation and phylogenies detected from isozyme markers in species of Lactuca. Hered. 77: 324-331. Kesseli, R.V., Paran, I., and Michelmore, R.W. 1990. Lactuca sativa. In Genetic maps. 5th ed. Edited by S.J. O'Brien. Cold Spring Harbor Press, N.Y. pp. 6.100-6.102. Kesseli, R. V., Paran, I., and Michelmore, R. W. 1991. The variation at RFLP loci in Lactuca spp. and origin of cultivated lettuce (L. sativa). Genome, 34: 430-436.
Lander, E.S., Green, P., Abrahamson, J., et al. 1987. MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics, 1: 174- 181. Landry, B.S., Kesseli, R., Farrara, B., and Michelmore, R.W. 1987a. A genetic map of lettuce (Lactuca sativa L.). with restriction fragment length polymorphism, isozyme, disease resistance and morphological markers. Genetics, 116: 33 1-337. Landry, B.S., Kesseli, R., Leung, H., and Michelmore, R.W. 19876. Comparison of restriction endonucleases and sources of probes for their efficiency in detecting restriction fragment length polymorphisms in lettuce (Lactuca sativa L.). Theor. Appl. Genet. 74: 646-653. Maeda, N., and Smithies, 0. 1986. The evolution of multigene families: human haptoglobin genes. Ann. Rev. Genet. 20: 81-108. Maquat, L.E., Chilcote, R., and Ryan, P.M. 1985. Human triosephosphate isomerase cDNA and protein structure. J. Biol. Chem. 260: 3748-3753. Marchionni, M., and Gilbert, W. 1986. The triosephosphate isomerase gene from maize: introns antedate the plant-animal divergence. Cell, 46: 133- 141. Michelmore, R.W., Hulbert, S.H., Landry B.S., and Leung, H. 1987. Towards a molecular understanding of lettuce downy mildew. In Genetics and plant pathogenesis. Edited by P.R. Day and G. J . Jelis. Blackwell Scientific Publications, Oxford. pp. 221-231. Noltmann, E.A. 1972. Aldose ketose isomerases. Enzymes, 1: 271-353. Odrzykoski, I. J., and Gottlieb, L.D. 1984. Duplications of genes encoding 6-phosphogluconate dehydrogenase in Clarkia (Onagraceae) and their phylogenetic implications. Syst. Bot. 9: 479-489. Paran, I., Kesseli, R.V., and Michelmore, R. W. 1991. Identification of restriction fragment length polymorphism and random amplified polymorphic DNA markers linked to downy mildew resistance genes in lettuce using near-isogenic lines. Genome, 34: 1021-1027. Pichersky, E. 1990. Nomad DNA-A model for movement and duplication of DNA sequences in plant genomes. Plant Mol. Biol. 15: 437-448. Pichersky, E., and Gottlieb, L.D. 1983. Evidence for duplication of the structural genes coding plastid and cytosolic isozymes of triose phosphate isomerase in diploid species of Clarkia. Genetics 105: 421-436. Pichersky, E., and Gottlieb, L.D. 1984. Plant triose phosphate isomerase isozymes. Plant Physiol. 74: 340-347. Pryor, T. 1987. The origin and structure of fungal disease resistance genes in plants. Trends Genet. 3: 157-161. Rogers, J.H. 1985. The origin and evolution of retroposons. Int. Rev. Cytol. 93: 187-279. Ryder, E. J., and Whitaker, T. W. 1976. Lettuce (Lactuca sativa). In Evolution of crop plants. Edited by N.W. Simmonds. Longman Scientific and Technical, Edinburgh. pp. 39-41. Sambrook, J., Maniatis, T., and Fritsch, F.F. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York. Sanger, F., Nicklen, S., and Coulsen, A.R. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74: 5463-5467. Shin, C.H., Lazar, G., and Goodman, H.M. 1986. Evidence in favor of the symbiotic origin of chloroplasts: primary structure and evolution of tobacco glyceraldehyde-2-phosphate dehydrogenases. Cell, 47: 73-80. Siracusa, L.D., Jenkins, N.A., and Copeland, N.G. 1991. Identification and application of repetitive probes for gene mapping in the mouse. Genetics, 127: 169-179. Soltis, P.A., Soltis, D.E., and Gottlieb, L.D. 1987. Phosphoglucose
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mutase gene duplications in Clarkia (Onagraceae) and their phylogenetic implications. Evolution, 41: 667-67 1. Tanksley, S.D., and Pichersky, E. 1988. Organization and evolution of sequences in the plant nuclear genome. I n Plant evolutionary biology. Edited by L.D. Gottlieb and S. Jain. Chapman and Hill, Inc., London. pp. 55-83.
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Wendel, J.F., Stuber, C.W., Goodman, M.M., and Beckett, J.B. 1989. Duplicated plastid and triplicated cytosolic isozymes of triosephosphate isomerase in maize (Zea mays L.). J. Hered. 80: 21 8-228.
