GENOMICS 14, 444-448 (1992)

Genetic Mapping of Tandemly Repeated Telomeric DNA Sequences in Tomato (Lycoper$iconesculentum) MARTIN W. GANAL, PIERREBROUN, AND STEVEND. TANKSLEY Department of Plant Breeding and Biometry, Come//University, 252 Emerson Hall, Ithaca, New York 14853 Received February 26, 1992; revised July 6, 1992

A telomere-associated tandemly repeated DNA seq u e n c e o f t o m a t o , T G R I, h a s b e e n u s e d to m a p t e l o meres on the tomato RFLP linkage map. Mapping was performed by monitoring the segregation of entire arrays of TGR I from a segregating F2 population using pulsed-field gel electrophoresis (PFGE). With this strate g y , f o u r t e l o m e r e s h a v e b e e n m a p p e d to t h e e n d s o f t h e short arm of chromosomes 9 and 12 and the long arms of chromosomes 5 and 11, using a saturated RFLP map of tomato containing approximately 1000 RFLP m a r k e r s . I n all f o u r c a s e s , t h e T G R I l o c u s m a p s to t h e end of the chromosome, and the distance between the most distal single-copy RFLP marker and the telomeric TGR I locus was between 1.6 and 9.6 cM. This indicates t h a t t h e r e g i o n c l o s e to t h e t e l o m e r e s d o e s n o t s h o w an e x c e s s i v e r a t e o f r e c o m b i n a t i o n c o m p a r e d to o t h e r r e g i o n s o f t h e g e n o m e a n d t h a t t h e R F L P m a p o f t o m a t o is essentially complete and covers the entire genome for all p r a c t i c a l p u r p o s e s . A d d i t i o n a l l y , t h e m a p p i n g t e c h n i q u e p r e s e n t e d h e r e s h o u l d b e g e n e r a l l y a p p l i c a b l e to the mapping of other tandemly repeated DNA sequences.

© 1992 Academic Press, Inc.

INTRODUCTION Genetic maps based on restriction fragment length polymorphisms (RFLPs) have now been constructed for a large number of organisms, such as mammals (DonisKeller et al., 1987), fungi (Hulbert et al., 1988; Tzeng et al., 1992), and plants (Bernatzky and Tanksley, 1986; Chang et al., 1988; Burr et al., 1988). Most of these maps are complete in the sense that they show as many linkage groups as there are chromosomes in the given organism. Assuming t h a t the used probes are randomly distributed throughout the genome and that genetic recombination is relatively equal along the chromosomes, any additional marker or trait should show linkage to at least one of the R F L P markers on such a map (Paterson et al., 1988). However, numerous data suggest t h a t genetic recombination can be highly reduced in regions around the centromeres and expanded in others. Because of this, a genetic map is not complete without information about the map position of sequences that 0888-7543/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

delimit each chromosome, i.e., the telomeres. Location of telomeres and/or telomere-associated sequences on a genetic map provides the ultimate proof that the entire genome is covered, and until this has been achieved, there is always the possibility that new genetic markers will expand the existing linkage groups. Unfortunately, the genetic mapping of telomeres is hindered by some intrinsic features of these sequences. Telomeres usually exist as long tandemly repeated arrays of a basic 7-bp oligonucleotide unit and, as part of their DNA replication mechanism, increase and decrease in length over generations (Zakian, 1989). Cloning telomeres has been hampered by the lack of restriction sites in such sequences and the presence of subtelomeric repeated DNA sequences. Only recently has part of t h a t problem been overcome by the use of a telomere complementation cloning system in yeast artificial chromosomes and other specialized cloning techniques (Riethman et al., 1989; de Lange et al., 1990). Nevertheless, this approach is complicated, and it is difficult to generate single-copy probes from such clones for mapping. In tomato, as in many other organisms (Zakian, 1989), chromosome ends are a complex of several different sequences: The most distal sequences of each chromosome are represented by a 7-bp repeat, TT(T/A)AGGG, which shows high sequence similarity to other published eukaryotic telomeres (Richards and Ausubel, 1988; Ganal et al., 1991). Separated by a sequence of yet unknown structure ranging from a few to more t h a n 100 kb in length is a tandemly repeated DNA sequence, T G R I (tomato genomic repeat I). T G R I is one of the most prominent repeated DNA sequences in tomato (Ganal et al., 1988). Its 77,000 copies are distributed over 27 sites, as shown by in situ hybridizations (Lapitan et al., 1989) of which 20 are associated with a telomere. Tomato (Lycopersicon esculentum) is one of the bestcharacterized plant systems. Its small genome size (approx 900,000 kb), agricultural value, and a large number of known mutants make it a model system for the characterization of agriculturally important genes. Genetic studies are facilitated by its propagation as self-pollinated highly inbred plant species. In the last several years, we have constructed a saturated genetic map of

444

MAPPING OF TELOMERIC DNA SEQUENCES t o m a t o (L. esculentum) b a s e d o n R F L P s . T h i s m a p c u r r e n t l y c o n t a i n s m o r e t h a n 1000 loci d i s t r i b u t e d a l o n g t h e 12 c h r o m o s o m e s a n d c o v e r s a t o t a l g e n e t i c d i s t a n c e o f 1276 c M ( T a n k s l e y et al., 1992). S u c h a h i g h n u m b e r of markers on a single map has been achieved for only a few o t h e r o r g a n i s m s t o d a t e . H o w e v e r , b e c a u s e o f t h e lack of information on the position of the telomeres, it still cannot be considered complete. Because of this, we h a v e e m p l o y e d p u l s e d - f i e l d gel e l e c t r o p h o r e s i s t o m a p telomere-associated sequences directly on this map. TGR I was used as a genetic marker to delimit some of the tomato linkage groups, and the data presented in this paper indicate that the telomeres of tomato are very tightly linked to the most distal RFLP markers. M A T E R I A L S A N D METHODS

Plant material. For RFLP mapping in tomato, the F2 generation of an interspecific cross between two inbred accessions, L. esculentum TA 55 × L. pennellii TA 56, was used (Bernatzky and Tanksley, 1986). This population originally consisted of 67 individuals, generated from a single F1 plant, of which 50 have been maintained permanently as shoot cultures. Young plants that were transferred to the greenhouse were used as a source of protoplasts in the DNA isolation procedure. DNA isolation from protoplasts. High-molecular-weight DNA from tomato protoplasts was isolated essentially as previously described (Ganal and Tanksley, 1989). Whereas L. esculentum TA 55 yielded approximately 1 X 107 protoplasts/g of leaf material, it was very difficult to obtain any stable protoplasts and high-molecularweight DNA of sufficient quality from L. pennellii. In fact, the yield of protoplasts and DNA quality varied from plant to plant and protoplast yield segregated as a quantitatively inherited trait in the F2 progeny of this cross. On average, the yield of protoplasts was 5 x 105-5 X 106 protoplasts/g of leaf material in individual progeny plants. Because of that, the final concentration for embedding was only 2 X 106 protoplasts/ml. Blocks with high-molecular-weight DNA were washed and digested as described (Ganal and Tanksley, 1989). Separation was performed on 1% agarose CHEF gels (Chu et al., 1986) using 0.5× TBE (1X TBE = 0.09 M Tris-borate, 2 mM EDTA, pH 8.3) as running buffer. After electrophoresis, the gels were transferred onto HyBond N + (Amersham) for hybridization. DNA probes and hybridization. Segregation of the TGR I satellite DNA repeat of tomato was monitored by hybridization with a cloned 162-bp repeat (Ganal et al., 1988; Schweizer et al., 1988). Hybridization was performed according to Bernatsky and Tanksley (1986). Probes were labeled using the random hexamer technique, and hybridizations with TGR I were washed to 0.5X SSC, 0.1% SDS at 65°C before they were exposed to X-ray films (Kodak XAR). Exposure times varied between a few and 24 h. Mapping. All DNA fragments were initially scored as presence or absence (dominant/recessive). For one locus both alleles could be identified, and they were subsequently scored for all three possible genotypes. The map position of each segregating DNA fragment was determined on the existing high-density RFLP map of tomato (Tanksley et al., 1992) using the Mapmaker program (Lander et al., 1987). In addition, the data were analyzed for the occurrence of double crossover in the regions of interest. RESULTS

Detection of Restriction F r a g m e n t L e n g t h P o l y m o r p h i s m Using T G R I A s Hybridization Probe W h e n h i g h - m o l e c u l a r - w e i g h t D N A o f t o m a t o is dig e s t e d w i t h E c o R V o r BglII, s e p a r a t e d o n h i g h - r e s o l u -

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FIG. 1. Analysis of polymorphism of TGR I on pulsed-field gels. High-molecular-weight DNA of the two parents was digested with the restriction enzymes EcoRV and BglII, separated on pulsed-field gels, blotted, and hybridized with a TGR I probe. Lanes are as follows: Lycopersicon pennellii DNA digested with BglII (lane 1) and EcoRV (lane 2). L. esculentum DNA digested with BglII (lane 3) and EcoRV (lane 4). Fragment sizes were determined using yeast chromosomes as markers. Separation conditions were 100 s pulse time, 125 mA constant current for 60 h. t i o n p u l s e d - f i e l d gels o v e r a w i d e size r a n g e , a n d h y b r i d i z e d w i t h T G R I, t h e n u m b e r o f D N A f r a g m e n t s c o r r e s p o n d s w e l l w i t h t h e n u m b e r o f loci ( a p p r o x 27) b a s e d o n in situ h y b r i d i z a t i o n ( L a p i t a n et al., 1989) i n d i c a t i n g t h a t e a c h D N A f r a g m e n t r e p r e s e n t s a n i n d i v i d u a l locus. T h e r e f o r e , i f t h e r e is p o l y m o r p h i s m b e t w e e n t w o p a r ents of a mapping population, it should be possible to use such DNA fragments for the mapping of individual a r r a y s o f T G R I, a s h a s b e e n s h o w n f o r t h e c e n t r o m e r i c a l p h a - s a t e l l i t e a r r a y s i n h u m a n ( W i l l a r d et al., 1986; W a y e et al., 1987; M a h t a n i a n d W i l l a r d , 1990). F i g u r e 1 shows a survey of the two parents of our standard F2 m a p p i n g p o p u l a t i o n (L. e s c u l e n t u m T A 55 a n d L. p e n nellii T A 56), w h i c h w a s u s e d t o c o n s t r u c t a h i g h - d e n s i t y R F L P m a p , w h e n h y b r i d i z e d w i t h T G R I. F o r b o t h E c o R V a n d BgIII, t h e r e is e x t e n s i v e l e n g t h p o l y m o r p h i s m o n p u l s e d - f i e l d gels b e t w e e n t h e t w o p a r e n t s . F o r E c o R V , t h e m a j o r i t y o f T G R I D N A f r a g m e n t s f r o m L. esculentum T A 55 is b e l o w 500 k b , a n d t h i s a c c e s s i o n a p p a r e n t l y l a c k s a n y D N A f r a g m e n t s l a r g e r t h a n 850 k b , w h e r e a s L. pennellii s h o w s a n u m b e r o f D N A f r a g m e n t s i n e x c e s s o f 850 k b , m a k i n g t h i s e n z y m e w e l l s u i t e d f o r m a p p i n g i n t h i s size r a n g e . F i n a l l y i t s h o u l d b e n o t e d t h a t a c o m p a r i s o n o f t h e L. e s c u l e n t u m T A 55 p a t t e r n t o t h e p r e v i o u s l y p u b l i s h e d p a t t e r n for t h e c u l t i v a r V F N T c h e r r y ( L a p i t a n et al., 1989) s h o w s t h a t t h e T A 55 D N A fragments are generally shorter than the DNA fragm e n t s o f t h e c u l t i v a r V F N T c h e r r y o r L. pennellii (see a l s o B r o u n et al., 1992).

Segregation Analysis of T G R I D N A F r a g m e n t s on Pulsed-Field Gels H i g h - m o l e c u l a r - w e i g h t D N A w a s e x t r a c t e d f r o m 42 individual plants of the mapping population, digested w i t h E c o R V a n d BglII, a n d s e p a r a t e d o n p u l s e d - f i e l d

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FIG. 2. F2 segregation pattern for some TGR I satellite arrays. DNA from individual F2 plants was isolated from protoplasts and digested with EcoRV, blotted, and hybridized with a TGR I probe. This gel shows a subset of 14 plants used for mapping. Arrows show the segregating DNA fragments that have been scored for mapping. "e" indicates L. esculentum-specific allele, "p" represents an L. penneUii-specific allele. Separation conditions: 2 min pulse time, 150 mA constant current, 60 h separation time. gels, and the segregation p a t t e r n of T G R I satellite D N A arrays was m o n i t o r e d by hybridization. A n u m b e r of the P F G E fragments hybridizing with T G R I were found to be segregating in this population. Figure 2 shows the segregation p a t t e r n of the larger T G R I D N A fragments using the restriction e n z y m e E c o R V . X2 tests of the patt e r n confirmed a 3:1 segregation ratio (presence versus absence of the respective fragment) for several of the scorable fragments. T h e multitude of hybridizing fragm e n t s a n d absence of a 3:1 segregation p a t t e r n due to p o t e n t i a l overlaps of T G R I fragments, however, excludes the scoring of all segregating D N A fragments. For this reason, we focused on only well-separated T G R I D N A fragments in the high-molecular-weight range, which satisfied the 3:1 segregation criteria. Additionally, the identification of the c o m p l e m e n t a r y allele from the o t h e r p a r e n t for a given D N A f r a g m e n t was not straightforward because of the complex segregation p a t t e r n . Therefore, each hybridizing D N A f r a g m e n t was scored as presence and absence and m a p p e d as d o m i n a n t / r e cessive m a r k e r o n t o the existing R F L P map. However, for one set o f T G R I D N A fragments (see Fig. 2), we were able to localize the alleles of b o t h parents. As expected in such a case, the allele from L. esculenturn as well as the one from L. pennellii m a p p e d to precisely the same place with no r e c o m b i n a t i o n between them. T h e r e f o r e , the data for this locus have been combined, resulting in the identification of all t h r e e genotypes for this locus. F o r all o t h e r loci, we were not able to identify b o t h alleles, indicating large differences in size between the respective D N A fragments (alleles) in the two parents. Due to the high variability of the p r o t o p l a s t yield and D N A quality in the analyzed plants, it was not possible to determine reliably the genotype (homozygous or heterozygous) of each b a n d b y dosage. T h e occurrence of large differences in the size of the T G R I fragments at individual loci is also confirmed by the locus for which b o t h alleles have been identified. In this case, the length of the T G R I D N A f r a g m e n t in the E c o R V digestion was a p p r o x i m a t e l y 850 kb for the L. esculentum allele and 1100 kb for the L. pennellii allele

(Fig. 2). Finally, some D N A fragments in this size range have also been scored on filters from BglII digestions, and the same scoring was obtained for the two scorable BglII fragments with sizes of 940 a n d 970 kb, indicating t h a t the scoring is reproducible with different enzymes and t h a t these BglII fragments r e p r e s e n t the same loci as the T G R I fragments of the same size generated with E c o R V (see Fig. 1). It was possible to map four T G R I arrays onto chromosomes 5, 9, 11, and 12, respectively (Fig. 3). T h e m o s t likely position for each locus of T G R I is at the t e r m i n a l position of a c h r o m o s o m e arm. In two cases the lod score was less t h a n 3 (chromosomes 9 and 12). B u t even in those cases, for any other position (i.e., closer to the centromere), at least one double crossover would be required. In all cases, we were n o t able to find any singlecopy R F L P marker, a m o n g more t h a n 1000 ( T a n k s l e y et al., 1992), t h a t was f u r t h e r distal from the c e n t r o m e r e (based on positive lod scores) t h a n the m a p p e d T G R I array. T h e genetic distances between the T G R I loci and the closest R F L P m a r k e r is between 9.6 cM on the long arm of c h r o m o s o m e 11 and 1.6 c M on the short arm of c h r o m o s o m e 9, indicating very tight linkage between the T G R I loci and the single-copy R F L P markers from the high-density R F L P map. T h e s e data are in a g r e e m e n t with the in situ hybridization data from L. esculentum (Lapitan et al., 1989). T h e in situ hybridization data show t h a t for chromosomes 5, 11, and 12, t h e r e are only sites at the end of each c h r o m o s o m e arm. For chromosome 9, three arrays were detected by in situ hybridization. Two of t h e m are on the long arm of t h a t chromosome. T h e m a p p e d locus, reported here, represents the single locus on the short arm of c h r o m o s o m e 9, since t h a t c h r o m o s o m e has been oriented with respect to the classical and cytogenetic map (Young et al., 1988).

DISCUSSION In an effort to evaluate the degree of completion of a high-density R F L P map of tomato, we have used the telomere-associated repeat T G R I as a m a r k e r to map some of the telomeres. For this, entire arrays of T G R I were separated by m e a n s of pulsed-field gel electrophoresis, a n d segregation analysis of polymorphic T G R I D N A fragments was p e r f o r m e d using an F2 population. T h e use of T G R I instead of the true telomeric r e p e a t is valid since these two sequences are in close physical p r o x i m i t y and together comprise the h e t e r o c h r o m a t i c telomere structure of the t o m a t o c h r o m o s o m e s (Ganal et al., 1991). Additionally, most T G R I arrays do not show the features of the true telomeres, such as increase or decrease in length over generations (Zakian, 1989), which makes the direct scoring of telomeric D N A fragm e n t s very difficult. E x t e n s i v e analysis of the inheritance of T G R I arrays of selfed plants shows t h a t the spontaneous m u t a t i o n rate of T G R I arrays is lower t h a n t h a t of the telomeric arrays and the p a t t e r n is stable during the p l a n t d e v e l o p m e n t (Broun et al., 1992; un-

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FIG. 3. Map position of the analyzed TGR I arrays on a saturated RFLP map of tomato. The map position of the four mapped TGR I arrays is indicated as SAT. Only the lod 3 framework map of the relevant chromosomes is shown for clarity. For the loci on chromosomes 9 and 12 the lod score was less than 3, and the region of ambiguity is indicated by a vertical line. However, it should be emphasized that for any other map position on chromosome 9 or 12, at least one double crossover would be required. The number of plants that were informative in the two- and multipoint analysis (i.e., they were scored for both the most distal RFLP marker of the respective chromosome and the TGR I locus) is for chromosome 5, 34 plants, for chromosome 9, 35 plants, for chromosome 11, 34 plants, and for chromosome 12, 33 plants. published results). For practical purposes, T G R I also results in a m u c h stronger hybridization signal due to its long t a n d e m l y repeated arrays. Finally, T G R I arrays display a very high rate of p o l y m o r p h i s m and a wide size range on pulsed-field gels, which make it ideal for genetic mapping (Broun et al., 1992). T h e mapping of four telomeric loci of T G R I onto the R F L P map shows clearly that the high-density R F L P map of t o m a t o ( T a n k s l e y et al., 1992) is essentially complete. T h e distance b e t w e e n the m o s t distal R F L P marker(s) and the telomeric T G R I arrays is between 1.6 and 9.6 cM, with an average distance of 5.1 cM. Assuming that this value is valid for all c h r o m o s o m e s , the total map length of the complete R F L P map of t o m a t o should increase by approximately 120 c M or 9.4% compared to the value based solely on single-copy R F L P markers. T h i s m a k e s the R F L P map of t o m a t o for all practical purposes complete and should allow a good coverage of the t o m a t o g e n o m e for the mapping of single-gene and polygenic traits (Paterson et al., 1988).

Additionally, with an average distance of 5.1 cM between the telomeres and the m o s t distal single-copy R F L P marker, telomeric regions do not likely display a greatly increased rate of recombination. T h i s is in agreem e n t with observations from the h u m a n R F L P map, where it was found that a telomeric position alone does not generally result in higher recombination values; rather, if there is a higher frequency of recombination, it is limited to specific "hot spots" (Donis-Keller et al., 1987; Hofker et al., 1990; Burmeister et al., 1991). Finally, segregation analysis on pulsed-field gel electrophoresis as described in this paper, could be used to map not only telomeric repeats, but any given t a n d e m l y repeated D N A sequence in the genome, provided it is polymorphic and within the size range of separation by PFGE, such as centromeric satellite repeats (Willard et al., 1986; W a y e et al., 1987; M a h t a n i and Willard, 1990). In this laboratory, it has been possible to use P F G E to genetically map t a n d e m l y repeated telomeric D N A sequences in rice (K. W u and S. D. Tanksley, unpublished

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