Proc. Nati. Acad. Sci. USA Vol. 89, pp. 1354-1357, February 1992 Telomeric arrays display high levels of heritable polymorphism among closely related plant varieties (hypervariability/pulsed-fleld gel electrophoresls//minisatelite) PIERRE BROUN, MARTIN W. GANAL, AND STEVEN D. TANKSLEY Department of Plant Breeding and Biometry, Cornell University, Ithaca, NY 14853 Communicated by Peter Starlinger, November 1, 1991 (received for review September 2, 1991)

ABSTRACT Tomato are composed ofa terminal in a mendelian fashion, these sequences might serve as 7-base-pair and a closely linked 162-base-pair genetic markers for telomeres. subtelomeric repeat (TGRI). Together, these repeats account for 2% of the total chromosomal DNA. Pulsed-field gel elec- trophoresis has been used to examine the long-range physical MATERIALS AND METHODS structure of these arrays in closely related varieties, and we Plant Material. Six tomato varieties were used in this report here that these arrays are undergoing heritable changes study: L. esculentum cvs. Rio Grande PtoR, San Marzano, at a frequency as great as 2% per generation. Moreover, TA55-VF36, UC82B, Vendor Tm2a, and VFNT Cherry. A comparisons with other known hypervariable probes (e.g., population of 20 progeny plants derived from a selfed inbred human and M13 sequences) revealed that telo- plant (L. esculentum cv. VFNT Cherry) was used to deter- meric sites are more variable than any other known region of mine a mutation rate per generation per fragment for both the plant genome and can be used to distinguih closely related TGRI and TEL. An F2 population consisting of45 plants from plant varieties (tomato and melon) that are otherwise very a cross between L. esculentum (TA55-VF36) x Lycopersicon similar at the DNA level. The fact that the polymorphisms are penneffli (TA56) was used for segregation analysis of the inherited in a mendelian fashion suggests applications in ge- TGRI fragments. Two melon varieties (Cucumis melo cvs. netic mapping of telomeres and identification of varieties. Honeydew and TAM Uvalde) were used to determine the level of polymorphism detected with TEL in this species. Self-pollinated plant species often contain less genetic vari- Pulsed-Field Gel Electrophoresis. High molecular weight ation than their outcrossing counterparts, making it more tomato DNA was isolated from protoplasts and digested with difficult to distinguish individuals and races even at the DNA Bgl II and EcoRV as previously described (15). Concentra- level (1-3). As a result, while it has been relatively easy to tions of 4 x 107 and 4 x 106 protoplasts per ml were used for identify unique DNA fingerprints for outcrossing crop spe- TEL and TGRI, respectively. The DNA fragments were cies such as maize and potato (4-6), it has been difficult to do separated on contour-clamped homogeneous electric field so for self-pollinated crops such as wheat, soybean, and (CHEF) gels (16) in 1% agarose. Separation conditions in- tomato (3, 7, 8). cluded a 60-sec pulse time and a 60-hr running time at 150 V, Recently we have identified and physically mapped two in 0.5x TBE (lx TBE = 0.09 M Tris borate, pH 8.3/2 mM tandemly repeated sequences in tomato (Lycopersicon escu- EDTA) at 12°C. Similarly, melon DNA was digested with lentum L.) that make up the outermost portion of the chro- Mlu I. The CHEF gel running conditions in this case were 24 mosomes (9, 10). Most tomato telomeres are composed of hr at 70-sec pulse time and 24 hr at 20-sec pulse time. three separate domains: (i) terminal 7-base-pair (bp) repeat Standard Electrophoresis. Tomato DNA was isolated as representing the telomere itself (denoted TEL; more than described (17) and cleaved with Hinfl, Bgl II, and EcoRV. 3000 copies per locus), which shows a high degree of homol- The digested DNA was separated on 0.8% agarose gels (18) ogy with both plant and animal telomeres (9, 11); (ii) a 162-bp or 1% agarose gels. subterminal satellite repeat (TGRI, 500-10,000 copies per Hybridization. Blotting was performed onto Hybond-N+ locus), which is found at 20 ofthe 24 telomeres and is specific membranes (Amersham) in 0.4 M NaOH. Hybridization to tomato and closely related species (10, 12); and (iii) a probes were the Arabidopsis telomeric repeat (11), the to- spacer region ofyet unknown structure that separates the two mato macrosatellite probe TGRI (9, 12), the 33.6 and 33.15 arrays and is a few kilobases (kb) to a few hundred kilobases human probes (18), and a set of 15 cloned in length (Fig. 1). low-copy-number tomato sequences used for RFLP mapping It has been speculated that telomeric tandem arrays such (3). Hybridization was performed as previously described (9, as TGRI and TEL are susceptible to unequal crossing-over 15) but in the absence of salmon testes DNA as a blocking between homologous or even nonhomologous chromo- agent in the case of human minisatellite probes. somes-a process likely to yield high polymorphism (13, 14). Computation of Genetic Distances Among Accessions. The The aim of this study was to test the level of polymorphism average proportion F of fragments in common between all detected by TGRI and TEL on tomato varieties that other- = wise show little variation at the DNA level. We report here pairs of accessions was determined for each probe: F that these sequences, when separated on pulsed-field gels, 2nXY/(nX + nY), where nX and nY are the number of are hypervariable, even in a self-pollinated crop such as fragments for varieties X and Y and nXY is the number of tomato. They also detect high polymorphism among melon fragments in common (19). A corresponding D value (D = 1 varieties. Moreover, since the polymorphisms are inherited - F) was used to estimate the genetic distance among all varieties. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: CHEF, contour-clamped homogeneous electric field; in accordance with 18 U.S.C. §1734 solely to indicate this fact. RFLP, restriction fragment length polymorphism. 1354 Downloaded by guest on September 27, 2021 Genetics: Broun et al. Proc. Natl. Acad. Sci. USA 89 (1992) 1355

TEL R - centromere _-- 20 - 50 kb ca. I 00 kb 100 -I 000 kb FIG. 1. Schematic structure of a tomato telomere. RESULTS AND DISCUSSION ofgenetic divergence, and since it is based on a per fragment calculation, it reduces bias that may result from using probes Level of Polymorphism Detected by TEL and TGRI. High that detect unequal numbers of fragments. molecular weight DNA from six tomato varieties was iso- The average D value for the low-copy-number RFLP lated, cut with the restriction enzymes Bgl II and EcoRV, and probes was 0.04 ± 0.02. M13 detected no clear differences separated on pulsed-field gels. Neither ofthese enzymes cuts among the varieties (D = 0.00). The 33.6 and 33.15 human in the TEL or the TGRI repeats. However, in most cases, minisatellite probes gave D values of 0.14 ± 0.06 and 0.28 ± they do cut in the spacer region separating these two se- 0.09, respectively, whereas TEL and TGRI had D values of quences and therefore allow the individual analysis of both 0.92 ± 0.09 and 0.65 ± 0.15. Values for TEL are arrays (9). The length of the fragments generated by these and TGRI enzymes should thus be roughly proportional to the number significantly higher than those ofthe other probes tested. On of copies of the TEL and TGRI repeat. As shown in Fig. 2 A the basis of these data we estimate that TEL and TGRI and B both probes revealed an extremely high level of detect, on average, 2.5 to 6.6 times more polymorphism than variation among tomato cultivars, and 10-20o of the TGRI the human minisatellite probes and at least 16 times more bands were cultivar specific. To compare this high level of variation than M13 or standard single-copy RFLP probes variation with other known sequences, DNA from the same (Fig. 3). varieties was cleaved with Bgl II, EcoRV, and Hinfl, sepa- Plant Development and Stability ofTGRI and TEL Patterns. rated on standard agarose gels, and hybridized with a variety To be useful in fingerprinting or genetic studies, TEL and of probes: 15 low-copy-number tomato restriction fragment TGRI must give reproducible results when tissues ofdifferent length polymorphism (RFLP) probes, previously found to be ages are sampled. In humans it has been observed that useful in the discrimination of tomato cultivars (3) and three telomeric repeats can decrease in length as cells grow older minisatellite probes (M13, 33.6, and 33.15) previously shown (23, 24). Therefore, we tested DNA isolated from leaves of to detect hypervariable regions in both animals and plants individual plants at different times during development, from (18, 20-22) (Fig. 2 C and D). The amount of variation per 4-week-old plants to plants that were more than 6 months old. fragment detected with each probe was determined by cal- Although we cannot rule out the occurrence of small changes culating the D value for all pairs of varieties. D is a measure in copy number that would not be detectable by pulsed-field gel electrophoresis, we did not observe any changes in the A B length of individual telomeric or TGRI arrays at different developmental stages (data not shown). We therefore con- k b kb clude that the length ofthe individual arrays oftelomeres and 690 780 TGRI repeats is generally stable during somatic development for the period tested. Mutability and Heritability of Telomeric Fragments. To oN a determine whether telomeric polymorphisms are stably I, transmitted from one generation to the next and to estimate * a per generation mutation rate, 20 selfed progeny were 450~~~f 4 50 obtained from a single inbred plant (cv. VFNT Cherry) and tested with the TGRI probe. A total of 400 fragments were

'* - scored in these progeny (20 plants x 10 fragments per plant 250 * ; 9.4 SW Mdw _M o 1 2 3 4 56 1.0 - 1 2 3 4 5 6 _F. 0 mea-8< 0 -- I u.q 0.8 - 0 c D 0 kb k b *, j 0.6 - 2. 2 3.1 * '* 2 3 4 5 501234 6 0 0 0.4 - 9.4 I C, b~ 0.2 - 6.6 . * 123456_ 0)

1. ----s.:.4

- 4.4 0.0 I ,-,-I . I I LCP 33-6 33-15 TGR I TEL Probe

12 34 56 FIG. 3. Level ofpolymorphism detected among cultivars with the different probes. Mean average D values (0 < D < 1) plus standard FiG. 2. Comparison of hybridization patterns of six tomato deviation from all pairwise comparisons, calculated for the genomic varieties with different probes. (A) Telomeric probe (TEL). (B) low-copy-number probes (LCP), the human minisatellites 33.6 and Macrosatellite TGRI probe. Both are hybridized to high molecular 33.15, TGRI, and TEL. The average values are significantly differ- size DNA separated on a CHEF gel. (C) Human minisatellite 33.15. ent, as shown by a two-way analysis of variance controlling for (D) A low-copy-number tomato clone, F 1292 (2). Lanes: 1, cv. Rio variety pairing (F = 254.28, P = 0.0001) and paired t tests adjusted Grande PtoR; 2, cv. San Marzano; 3, cv. TA55-VF36; 4, cv. UC82B; for multiple comparison by using the Bonferroni method. The 5, cv. Vendor Tm2a; 6, cv. VFNT Cherry. obtained t values were highly significant (P < 0.0001). Downloaded by guest on September 27, 2021 1356 Genetics: Broun et al. Proc. Natl. Acad. Sci. USA 89 (1992)

x 2 alleles). Two novel fragments were observed in each of kb five plants (Fig. 4A). The 10 observed changes involved the same two fragments in each mutant plant, suggesting either that some of the TGRI macrosatellites are more susceptible 580 to change than others or that the observed mutations are derived from a single mutant sector in the parent plant. The I0 .4'a mutation rate per fragment per generation is thus estimated 450 to be 10/400 or 2.5% if each novel band arises from an independent event, and 2/20 (2 fragments out of 20 scored in WO- 360 the parent) or 10% if the changes are derived from a mutant sector. These figures are comparable to the highest mutation rates reported for minisatellites in humans and other orga- nisms (25). Ten progeny plants were also screened for TEL 250 variation. Six fragment changes were detected from a total of 80 fragments examined (7.5%). This suggests a mutation rate for TEL that is at least as high as that estimated for TGRI. The above experiment establishes that inbred lines trans- mit their macrosatellite patterns from one generation to the next (albeit with a high mutation rate), but it does not address 1 2 the question of segregation for telomeric polymorphisms. To FIG. 5. Two melon cultivars (C. melo) compared for their telo- investigate this issue, we monitored the segregation of TGRI meric patterns detected with TEL. Lanes: 1, cv. Honeydew; 2, cv. and TEL polymorphisms in an F2 population derived from a TAM Uvalde. cross between two tomato species (L. esculentum x L. pennel ii). The parents differed for the length of most TGRI the tomato RFLP map (M.W.G., P.B., and S.D.T., unpub- and TEL arrays. Because of the large number of segregating lished data). fragments, it was not possible to monitor all polymorphisms. Polymorphism of TEL in Melon. From the previous obser- We thus focused on six telomeric fragments detected with the vations, the question arises whether the hypervariability TGRI probe. Forty-five plants were tested and all fragments observed for tomato telomeric arrays also occurs in other segregated in a ratio consistent with the expected 3:1 (0.05 < plant species. To shed some light on this question, a com- P < 0.97), indicative of monogenic inheritance (Fig. 4B). parison was made between two varieties of melon (Cucumis Moreover, most fragments segregated independently from melo). Melons were chosen because they have previously one been shown to differ very little at the DNA level (4). High another and map to the end ofdifferent linkage groups on molecular weight DNA was isolated, cut with various re- striction enzymes, and separated on a pulsed-field gel. The kb TEL probe hybridized strongly to melon DNA and allowed A , 40 4* 690 clear differentiation of the two cultivars, suggesting that 41, hypervariability may also be a feature of telomeres from this species and possibly other plant species (Fig. 5). The level of polymorphism for the telomere and the sub- a 450 telomeric TGRI repeat in tomato is much higher than that reported thus far for any other plant sequence. These results b. and the variability observed in melon confirm the hypervari- able nature of telomeric repeats in plants, as has been I*I reported in mice (26, 27). In addition, the current study shows 360 that associated subtelomeric sequences such as TGRI are also hypervariable. These sequences might therefore be useful in distinguishing plant varieties even in inbreeding P 1 2 3 4 species, which usually display very low polymorphism at the DNA level and cannot be discriminated otherwise. The fact kb that most of these polymorphisms are inherited in a mende- lian fashion suggests that they may be useful as genetic B a 81 0 markers, for locating the virtual ends ofgenetic linkage maps b- 580 or for localizing genes (including quantitative trait loci) near C - the ends of chromosomes. Elucidation of the mechanism(s) responsible for the extremely high polymorphism in telo- meric regions awaits further investigation. Aa .. *I 4 5 0 We thank Drs. C. Aquadro, M. Bonierbale, and G. Martin for their It# critical reading ofthe manuscript. This work was supported by U.S. Department of Agriculture Competitive Grant 88-37262-3921 and FIG. 4. Genetic transmission of the TGRI telomeric fragments. U.S./Israel Binational Agricultural Research and Development (A) Mutability. Selected plants from the progeny of a selfed VFNT Fund Grant US-1388-87 to S.D.T.; P.B. was supported by a fellow- cherry plant are shown for their TGRI patterns. Lanes: P, parent; 1, ship from the Biocem Company (Limagrain group). Additional 2, and 3, progeny plants displaying a novel band not found in P; 4, support was provided by the Cornell National Science Foundation progeny plant displaying the parental pattern. Arrowheads a and b Plant Science Center, a unit in the U.S. Department of Agriculture- indicate the new bands observed in the progeny plants. (B) Segre- Department of Energy-National Science Foundation Plant Science gation of TGRI bands among selected plants from an F2 mapping Centers Program and a unit of the Cornell Biotechnology Program, population (L. esculentum TA55 x L. pennelffi). The bands a, b, and which is sponsored by the New York State Science and Technology c gave the following ratios and X2p values (for goodness offit to 3:1): Foundation, a consortium ofindustries, and the U.S. Army Research a, 26:10 (P > 0.995); b, 22:14 (P > 0.1); c, 25:10 (P > 0.995). Office. Downloaded by guest on September 27, 2021 Genetics: Broun et al. Proc. Natl. Acad. Sci. USA 89 (1992) 1357

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