High density molecular linkage maps of the tomato and potato genomes.

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<:opyright 0 1992 by the Genetics Society of America

High Density Molecular Linkage Maps of the Tomato and Potato Genomes

S. D. Tanksley, M. W. Ganal, J. P. Prince, M. C. de Vicente, M. W. Bonierbale, P.

Broun,

T. M. Fulton,

J. J.

Giovannoni,

S. Grandillo, G. B. Martin, R. Messeguer,

J. C. Miller, L. Miller,

A.

H. Paterson, 0. Pineda, M. S. Riider, R. A. Wing, W. Wu and N. D. Young

Department of Plant Breeding and Biometry, Cornell University, Ithaca, New York 14853

Manuscript received March 23, 1992 Accepted for publication August 28, 1992

ABSTRACT

High density molecular linkage maps, comprised of more than 1000 markers with an average spacing between markers of approximately 1.2 cM (ca. 900 kb), have been constructed for the tomato and potato genomes. As the two maps are based on a common set of probes, it was possible to determine, with a high degree of precision, the breakpoints corresponding to 5 chromosomal inversions that differentiate the tomato and potato genomes. All of the inversions appear to have resulted from single breakpoints at or near the centromeres of the affected chromosomes, the result being the inversion of entire chromosome arms. While the crossing over rate among chromosomes appears to be uniformly distributed with respect to chromosome size, there is tremendous heterogeneity of crossing over within chromosomes. Regions of the map corresponding to centromeres and centromeric heterochromatin, and in some instances telomeres, experience up to 10-fold less recombination than other areas of the genome. Overall, 28% of the mapped loci reside in areas of putatively suppressed recombination. This includes loci corresponding to both random, single copy genomic clones and transcribed genes (detected with cDNA probes). The extreme heterogeneity of crossing over within chromosomes has both practical and evolutionary implications. Currently tomato and potato are among the most thoroughly mapped eukaryotic species and the availability of high density molecular linkage maps should facilitate chromosome walking, quantitative trait mapping, marker- assisted breeding and evolutionary studies in these two important and well studied crop species.

I

N 1980 BOTSTEIN et al. proposed the construction of a genetic linkage map in humans based on

restriction fragment length polymorphisms (RFLPs) (BOTSTEIN et al., 1980). The success of their idea has been verified by the fact that RFLP linkage maps have already been constructed, not only for humans, but f o r a wide variety of other organisms (see O’BRIEN 1990). Most of the RFLP maps published to date have been of low or moderate density (ie., average marker spacing >5 cM) and have included 50-300 markers. While these maps are useful tools for many genetic endeavors, they have inherent limitations that would

be overcome by the development of high density maps in which the markers are spaced at very close intervals throughout the genome.

High density maps can serve a number of purposes in basic and applied research. First, they a r e a key

tool for chromosome walking. In order to clone a

g e n e by chromosome walking, it is necessary to iden- tify molecular marker(s) closely linked to the gene of interest to provide a starting point for t h e walk (WICK- ING a n d WILLIAMSON 1991). High density linkage maps can provide starting points for chromosome walks to virtually any gene in the genome. Second, high density maps have direct application in plant and animal breeding since they virtually assure that any gene of interest will be tightly linked t o a t least o n e

(kwrtics 132: 1141-1 160 (Drcenlbrr, 1992)

molecular marker. Such tight linkages can be ex-

ploited for marker-based selection of desirable genes in breeding programs (BURR et al. 1983; TANKSLEY et al. 1989). Finally, high density linkage maps provide

a greater probability that the entire genome is com- pletely covered with molecular markers (ie., there are no large, markerless gaps in the map). This last point is especially important when using molecular linkage maps to detect and characterize loci underlying quantitative traits where one needs to be assured that the entire genome has been uniformly surveyed (PATER- SON et al. 1988; LANDER and BOTSTEIN 1989).

To facilitate map-based cloning in crop plants and establish a general tool for marker-enhanced breed-

ing, we have constructed a high density map of t h e tomato/potato genomes comprised of more than 1000

markers (mainly RFLPs). A number of morphological a n d isozyme markers have also been mapped with respect to RFLP markers making it possible to orient the molecular linkage map with both the classical morphological and cytological maps of tomato. This

level of marker saturation offers new opportunities for genome research in tomato and potato and reveals a number of interesting features of chromosome struc- ture and evolution that were not apparent from pre-

vious linkage maps of the tomato/potato genomes

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1142 S. D. Tanksley et al.

MATERIALS AND METHODS

Tomato: A population of 67 F2 plants derived from the interspecific cross Lycopersicon esculentum cv VF36-Tm2a X Lycopsersicon pennellii LA7 16 served as the mapping population for tomato. The two species used in this cross have the same chromosome constitution (2n = 2x = 24) and all evidence suggests that their genomes are homosequential (KHUSH and RICK 1963). Moreover, interspecific hybrids between the two species are highly fertile and demonstrate near normal levels of meiotic pairing and crossing over (KHUSH and RICK 1963; RICK 1969).

Potato: A population of 155 plants, derived from the cross Solanum tuberosum (2x = 24) X Solanum berthaultii backcrossed to S. berthaultii, constituted the mapping population for potato. S. tuberosum and S. berthaultii are closely related taxonomically and were previously known to give rise to fertile hybrids (MEHLENBACHER, PLAISTED and TIN-

GEY 1983).

Probes and markers: Random single and low copy RFLP probes were obtained from three cDNA libraries [CD = derived from total mRNA from tomato leaves, (BERNATZKY and TANKSLEY 1986); CT = derived from mRNA from tomato epidermal tissue (Yu et a l . , in preparation); CP = derived from total mRNA of potato (GEBHARDT et al. 1989)] and four genomic libraries [TG = derived from PstI or GcoRI size-selected tomato genomic fragments (MILLER and TANKSLEY 1990); size-selected sheared tomato genomic clones (ZAMIR and TANKSLEY 1988); GP = PstI size-selected potato genomic fragments (GEBHARDT et al. 1989)l. A number of clones corresponding to known genes were also used as probes for RFLP mapping and are listed in Table 1. Probes maintained at Cornell University are available upon request.

Methods used for probe preparation, Southern hybridi- zation and autoradiography were the same as previously published with the exception that clone inserts were polym- erase chain reaction (PCR) amplified before radiolabeling (BERNATZKY and TANKSLEY 1986).

Isozyme markers segregating in the tomato population were scored using previously described methods (VALLEJOS

1983). The positions of morphological markers on the molecular map (including many disease resistance genes) were determined from previously published work or were obtained by direct mapping against RFLP markers in segregating populations (see Table 1 for references).

Map construction: All autoradiographs from segregating populations were scored at least twice by different individ- uals. Ambiguous genotypes were treated as missing data for map construction. Both the tomato and potato maps were constructed using MapMaker software on a Sun I1 worksta- tion (LANDER et al. 1987). All pairs of linked markers were first identified using the ‘group’ command LOD > 5, RF = 0.20. Cosegregating markers (e.g., markers showing no recombination with one another) were identified by scanning two-point linkage data. Framework maps were constructed using only one marker from each set of cosegregating markers. The “orders” command was used to establish the framework order of markers within groups and the “ripples” command was used to verify the order. Markers were re- tained within the framework map only if the LOD value for ripples was >3 (probability of deduced sets of triplet orders 1000 times more likely than alternative orders). All remain- ing markers were assigned to intervals within the LOD 3 framework using the “try” command. Map units (centimor- gans, cM) between markers were calculated using the KO-

SAMBI (1 944) function. The chromosomal affiliation of each linkage group was established by identifying, within each group, markers of known chromosomal position based on

previously published work (BERNATZKY and TANKSLEY 1986).

RESULTS AND DISCUSSION

Tomato map

Marker distribution: A total of 1030 molecular markers were mapped onto the tomato genome and together these markers cover 1276 map units (Figure

1). T h e haploid DNA content of tomato is estimated t o be approximately 950 Mbp (ARUMUGANATHAN and EARLE 1991) which means that, on average, 1 cM equals approximately 750 kb. However, this is only an average value a n d probably varies tremendously depending on which portion of the map is being considered (see next section). While each chromosome has at least 50 markers, the markers are not uniformly distributed across the chromosomes (Figure 2). The

tomato chromosomes differ considerably in size (and presumably DNA content) and these differences may account for much of t h e differential distribution of

markers. For example, the three largest chromosomes (chromosomes 1-3) together contain 38% of the markers, whereas the three smallest chromosomes (chromosomes 10-12) contain only 18% (Figures 1 a n d 2). A plot of the pachytene length of each chromosome us. the number of total markers per chromosome reveals a tight association between these two parameters ( r = 0.95, Table 2). The length of mitotic metaphase chromosomes is also highly correlated with the number of markers per chromosome ( r = 0.90) (Table

2).

T h e only chromosome for which t h e marker content is not well predicted by the chromosome length is chromosome 5 (Figure 2). Chromo- some 5 is the fifth largest chromosome according to measurement of pachytene chromosomes (BARTON

1950), yet it ranks 10th in terms of its marker content a n d has substantially fewer markers than chromosome

6 which has a pachytene length nearly identical t o chromosome 5 (Figure

2).

RAMANNA and PRAKKEN (1967) proposed reclassifying chromosome 5 as one of the shortest chromosomes (also based on pachytene a n d somatic lengths). Similar conclusions were

reached by SHERMAN and STACK (1 992) based on two- dimensional spreads of synaptonemal complexes. T h e results presented here also support this proposal.

T h e relative number of loci corresponding to pu-

tative coding regions ( i e . , loci detected with cDNA probes) us. that corresponding to random genomic clones also differs considerably from chromosome t o chromosome (Figure 2). For example, the largest chromosomes (1-3) have more total markers but a lower proportion of loci corresponding to cDNAs than do the smaller chromosomes (Figure 2). A chi- square contingency test reveals that the differences in proportion of cDNA markers among the different chromosomes are significant ( P = 0.02).

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Tomato/Potato Linkage Maps 1143

TABLE 1

Genes of known function or phenotype that have been mapped onto the molecular map of tomato/potato (morph morphological marker) Gene 6Pgdh-1 6Pgdh-2 6Pgdh-3 a ae af ag alb ACCl ACC2 ACC3 ACC4 ACO-I ACO-2 Adh-1 Adh-2 Afs-I A@-2 B

CAB I

CAB2 CAB3 CAB4 CAB5 CAB6 CAB7 CAB8 CAB1 I

CAB12 cf-2 Cf-9 CHSl CHS3 CHS4 E4 E8A E8B Est-1 Est-2 Est-4 Est-5 Est-6

Type Product/phenotype Chromosome Reference

Isozyme 6-Phosphogluconate dehydrogenase Isozyme 6-Phosphogluconate dehydrogenase Isozyme 6-Phosphogluconate dehydrogenase Morph Anthocyaninless

Morph Entirely anthocyaninless Morph Anthocyanin free Morph Anthocyanin gainer Morph Albescent

RFLP ACC synthase RFLP ACC synthase RFLP ACC synthase RFLP ACC synthase Isozyme Aconitase Isozyme Aconitase

Isozyme Alcohol dehydrogenase Isozyme Alcohol dehydrogenase Isozyme Acid phosphatase Isozyme Acid phosphatase Morph Beta carotene

RFLP Chlorophyll u/b binding polypeptide RFLP Chlorophyll a/b binding polypeptide RFLP Chlorophyll a / b binding polypeptide RFLP Chlorophyll a / b binding polypeptide RFLP Chlorophyll a/b binding polypeptide RFLP RFLP RFLP RFLP RFLP Morph Morph RFLP RFLP RFLP RFLP RFLP RFLP Isozyme

Chlorophyll a / b binding polypeptide Chlorophyll a / b binding polypeptide Chlorophyll a / b binding polypeptide Chlorophyll a / b binding polypeptide Chlorophyll a / b binding polypeptide Resistance to Cladosporium fulvum Resistance to Cladosporium fulvum Chalcone synthase

Chalcone synthase Chalcone synthase

Ethylene inducible polypeptide Ethylene inducible polypeptide Ethylene inducible polypeptide Esterase

Isozyme Esterase Isozyme Esterase Isozyme Esterase Isozyme Esterase

4 12 5 I 1 8 5 10 1 2 8 I 2 5 12 7 4 6 6 8 6 2 8 3 7 12 5 10 10 6 3 6 I 6 5 9 3 9 3 2 9 1 2 2 2

TANKSLEY and KUEHN (1985); BERNATZKY and TANKSLEY TANKSLEY and KUEHN (1 985)

TANKSLEY and KUEHN (1 985); BONIERBALE, PLAISTED and RICK (1980); S. GRANDILLO and S. D. TANKSLEY (unpub- RICK (1980); S. GRANDILLO and S. D. TANKSLEY (unpub- RICK (1 980); G. B. MARTIN and S. D. TANKSLEY (unpub- RICK (1980); S. GRANDILLO and S. D. TANKSLEY (unpub-

RICK (1980); S. GRANDILLO and S. D. TANKSLEY (unpub- ROTTMAN et al. (1991)

ROTTMAN et al. (1991) ROTTMAN et al. (1991) ROTTMAN et al. (1 99 1)

TANKSLEY and RICK (1980); BERNATZKY and TANKSLEY TANKSLEY and RICK (1980) BERNATZKY and TANKSLEY

TANKSLEY and RICK (1 980); BERNATZKY and TANKSLEY TANKSLEY and JONES (1 98 1); this report

TANKSLEY and RICK ( 1 980); this report

TANKSLEY and RICK (1 980); this report

RICK (1980); S. GRANDILLO and S. D. TANKSLEY (unpub- VALLEJOS, TANKSLEY and BERNATZKY (1 986); BERNATZKY VALLEJOS, TANKSLEY and BERNATZKY (1 986); BERNATZKY VALLEJOS, TANKSLEY and BERNATZKY (1986); BERNATZKY PICHERSKY et al. (1987a); this report

PICHERSKY et al. (1987a); S. D. TANKSLEY, unpublished PICHERSKY et al. (1987b); this report

PICHERSKY et al. (1 988); this report PICHERSKY et al. (1 989); this report SCHWARTZ et a1 (1991)

SCHWARTZ et al. (1991)

JONES, DICKINSON and JONES ( 199 1)

JONES, DICKINSON and JONES (1991)

A. DREWS and R. GOLDBERG, personal communication A. DREWS and R. COLDBERG, personal communication A. DREWS and R. GOLDBERG, personal communication LINCOLN et al. (1 987); this report

LINCOLN et al. (1 987); this report LINCOLN et al. (1987); this report

TANKSLEY and RICK (1 980); BERNATZKY and TANKSLEY TANKSLEY and RICK (1 980); this report

TANKSLEY and RICK (1 980); BERNATZKY and TANKSLEY TANKSLEY and RICK (1980); BERNATZKY and TANKSLEY TANKSLEY and RICK (1 980); BERNATZKY and TANKSLEY

(1 986)

TANKSLEY (1 988) lished data) lished data) lished data) lished data) lished data) (1 986) (1 986) (1 986) lished data)

and TANKSLEY ( 1986)

and TANKSLEY (1 986) and TANKSLEY (1986)

data

(1 986)

(1986)

(4)

et al.

TABLE 1-Continued

Gene Type Product/phenotype Chromosome Reference

Est-7 Got-1 Got-2 Got-? h hl HMG2 HMG3 HOX7A HOX7B HOX7C HSF8 HSF24 HSF3O hY 1-2 1-3 ldh-1 j L-2 Mdh-3 Mi nor N r PGAL Pgi- 1 Pgm- 1 Pgm-2 PPO Prx- 1 PrX-2 Prx-3 Prx-7 PTC PTN Pto rin R45s R5s RBCSl Isozyme Isozyme Isozyme Isozyme Morph Morph RFLP RFLP RFLP RFLP RFLP RFLP RFLP RFLP Morph Morph Morph Isozyme Morph Morph Isozyme Morph Morph Morph RFLP Isozyme Isozyme Isozyme RFLP Isozyme Isozyme Isozyme Isozyme RFLP RFLP Morph Morph RFLP RFLP RFLP Esterase

Glutamate oxyaloacetate transami- Glutamate oxyaloacetate transami- Glutamate oxyaloacetate transami-

nase Hairs absent Hairless

HMG CoA reductase HMG CoA reductase Homeobox gene (tomato) Homeobox gene (tomato) Homeobox gene (tomato) Heat shock transcription factor Heat shock transcription factor Heat shock transcription factor Homogeneous yellow

Resistance to Fusarium oxysporum

Resistance to F. oxysporum race 3

Isocitrate dehydrogenase Jointless

Lutescent-2

Malate dehydrogenase

Resistance to root know nematodes Nonripening Never ripe Polygalaturonidase Phosphoglucoisomerase (cytosolic) Phosphoglucomutase (plastid) Phosphoglucomutase (cytosolic) Polyphenol oxidase Peroxidase Peroxidase Peroxidase Peroxidase Phytochrome

Patatin (tuber storage protein) Resistance to Pseudomonase syrin- Ripening inhibitor

45s ribosomal RNA

5 s ribosomal RNA

ss ribulose bisphosphate carboxyl- nase nase race 2 gae ase 2 4 7 7 10 11 2 3 2 2 10 2 8 8 10 11 7 1 1 1 10 7 6 10 9 10 12 ? 4 8 1 2 2 3 10 8 5 5 2 1 2

TANKSLEY and RICK (1980); BERNATZKY and TANKSLEY TANKSLEY and RICK (1980)

TANKSLEY and RICK (1980); BERNATZKY and TANKSLEY TANKSLEY and RICK (1 980); BERNATZKY and TANKSLEY

RICK (1980); S. GRANDILLO and S. D. TANKSLEY (unpub- RICK (1 980); KINZER, SCHWAGER and MUTSCHER (1 990)

J. NARITA and W, GRUISSEM (in preparation)

J. NARITA and W. GRUISSEM (in preparation) K. SCHARF (personal communication) K. SCHARF (personal communication)

K. SCHARF (personal communication) SCHARF et al. (1990)

SCHARF et al. (1990) SCHARF et al. (1990)

RICK (1 980); KINZER, SCHWAGER and MUTSCHER (1 990); S.

SAFARTTI et al. (1 989)

BOURNIVAL VALLEJOS and SCOTT (1989): TANKSLEY and BERNATZKY and TANKSLEY (1 986)

RICK (1980); R. A. WING and S. D. TANKSLEY (unpub- RICK (1980); S. GRANDILLO and S. D. TANKSLEY (unpub-

S. D. TANKSLEY (unpublished data) MESSEGEUR et al. (1 99 1)

RICK (1 980); J. GIOVANNONI and S. D. TANKSLEY (unpub- RICK (1980); J. GIOVANNONI and S. D. TANKSLEY (unpub- KINZER, SCHWACER and MUTSCHER (1990); this report TANKSLEY and RICK (1 980); BERNATZKY and TANKSLEY BERNATZKY and TANKSLEY (1986)

TANKSLEY and RICK (1980); BERNATZKY and TANKSLEY

S. NEWMAN (submitted)

TANKSLEY and RICK (1980); BERNATZKY and TANKSLEY

TANKSLEY and RICK (1980); BERNATZKY and TANKSLEY TANKSLEY and RICK (1 980); BERNATZKY and TANKSLEY

TANKSLEY and RICK (1 980); BERNATZKY and TANKSLEY

LISSEMORE, COLBERT and QUAIL (1 987)

BONIERBALE, PLAISTED and TANKSLEY (1 988); CANAL et al.

MARTIN, WILLIAMS and TANKSLEY (1 99 1)

J. GIOVANNONI and S. D. TANKSLEY (unpublished data) VALLEJOS, TANKSLEY and BERNATZKY (1986); BERNATZKY LAPITAN, CANAL and TANKSLEY ( 199 1)

VALLEJOS, TANKSLEY and BERNATZKY (1 986); BERNATZKY (1986)

(1 986)

GRANDILLO and S. D. TANKSLEY (unpublished data)

COSTELLO (1 99 1)

lished data) lished data) lished data) lished data) (1986) (1 986) (1986)

(1 986) (1 986) (1986)

(1990)

and TANKSLEY (1 986)

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Tomato/Potato Linkage Maps

TABLE 1-Continued

1145

Gene Type Product/phenotype Chromosome Reference

RBCS2

RBCS3

Skdh-1 Sm Sod-2

S P

SPa

!f

T311 Tm- 1 Tm-Za TOM25A TOM25B Tpi-2

U

Ve

wx

Y

RFLP

Isozyme Morph Isozyme Morph Morph

Morph

RFLP Morph Morph RFLP RFLP lsozyme

Morph

Morph RFLP Morph

ss ribulose bisphosphate carboxyl-

Shikimic acid dehydrogenase Resistance to Stemphilium Superoxide dismutase Self-pruning Sparsa

Trifoliate

5' patatin class I promoter Resistance to tobacco mosaic virus Resistance to tobacco mosaid virus Ripening related

Ripening related

Triose phosphate isomerase

Uniform ripening

Resistance to Verticillium Waxy

Yellow flesh (potato) ase

ase

3

2

I I 1

1 6 8

5

3

2

9 6 6

4

IO

7

8

3

VALLEJOS, TANKSLEY and BERNATZKY ( 1 986); BERNATZKY

VALLEJOS, TANKSLEY and BERNATZKY (1986); BERNATZKY

BERNATZKY and TANKSLEY ( 1 986) BEHARE et al. ( 199 1 )

D. ZAMIR (unpublished data) PATERSON et al. (1988)

RICK ( 1 980); S. GRANDILLO and S . D. TANKSLEY (unpub-

RICK (1980); G . B. MARTIN and S. D. TANKSLEY (unpub-

CANAL, LAPITAN and TANKSLEY ( 1 99 1 ) LEVFSQUE et at. (1990)

YOUNG et a f . (1988)

KINZER, SCHWACER and MUTSCHER (1990) KINZER, SCHWAGER and MUTSCHER (1990)

TANKSLEY and RICK ( 1 980), BERNATZKY and TANKSLEY

(1 986)

PATERSON et al. (1988); KINZER, SCHWAGER and MUTSCHER (1990)

JUVICK, BOLKAN and TANKSLEY ( 1 991) GEBHARDT et al. (1989)

BONIERBALE, PLAISTED and TANKSLEY (1988) and TANKSLEY ( 1 986)

and TANKSLEY (1 986)

lished data)

References are for mapping of loci onto molecular linkage map and/or source of probe used for such mapping.

units per chromosome is tightly correlated with both number of markers per chromosome and pachytene length ( r = 0.84 and 0.89, respectively) (Table 2). To test the nature of the relationships among these variables, slopes from linear regressions were calculated for normalized plots between pachytene length and cM/number of markers per chromosome. T h e slope for pachytene length us. cM/chromosome was 0.89 &

0 . 1 5 and the slope for pachytene length us. number of markers/chromosome was 0.95 2 0.10. T h e fact that both of these slopes are very close to 1 .O indicates that, in general, as the pachytene length of the chromosomes increases, the number of map units and number of markers increases in direct linear proportion. These findings are in contrast with earlier studies i n tomato which suggested a nonlinear relationship between the cytological and genetic maps (KHUSH and KICK 1968). The reason for this discrepancy is un- known but may be due to the fact that the earlier analyses were based on incomplete genetic maps-a variable of uncertainty acknowledged by authors of that earlier work.

Heterogeneity of marker density along the genetic linkage map: While the number of markers among

linkage groups appears to be uniformly distributed according to chromosome size, the distribution of map units within chromosomes varies dramatically, depending on which part of the linkage group is being ex-

amined (Figure 1). Good examples are chromosomes

3 and 12 where a preponderance of markers occur toward the middle of the linkage groups (Figure 1). To better evaluate the heterogeneity of marker density along the map, a series of histograms were pro- duced in which linkage maps for each chromosome were divided into 2-cM segments and the density of markers (per 2-cM interval) were plotted along the chromosome (see Figure 3 for sample histograms). For these plots, only those markers positioned at a LOD

>

3 were included. Based on these histograms, regions of high marker density could be identified in all chromosomes and a comparison with the pachytene karyotype of each chromosome suggests that the regions of high marker density correspond to centromeric areas and, in some instances, telomeric regions (Figure 3). In some cases the changes in marker density are sudden and dramatic. For example, the average density for most regions of chromosome 3 is one marker per 2-cM interval. However, in a region of approximately 10 cM near the center of the chromosome, the marker density increases more than 10-fold, to as high as 16 markers per 2-cM interval (Figure 3). Another good example is chromosome 4 (Figure 3) and similar, but less striking, changes in marker density could be seen for each of the other chromosomes (histograms not shown).

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S. D. Tanksley et al.

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FIGURE 1 .-Molecular linkage map of the tomato genome (left) and comparison with classical map (center) and cytological (pachytene) nap (right). Loci by tick marks on molecular map have been ordered with LOD > 3. Loci in bold correspond to known genes (see Table 1

fix details). Loci following commas cosegregate. Markers enclosed in parentheses have been located to corresponding intervals with LOD C 3. Position of undellined loci approximated from placement on previously published maps. All other loci mapped directly on FP population

o f 67 plants from L. esculentum X L. pennellii (see MATERIALS AND METHODS for details). Approximate positions of selected markers (bold)

(7)

Tomato/Potato Linkage Maps

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I v i t l l getlontic c-lo~tes ;uld c.I)XA clones for each of the tot11ato ~~lll~olllosollle~.

T A B L E 2

Correlation coefficients ( r ) for pairwise combinations among various chromosomal/map variables in tomato

c D N A Gcnotnicr Total Pachytcnr Mitotic

c. 51 0.7 I 0.83 0.84 0.89 0.91

cDNA 0.74 0.88 0.79 0.71

(;enotnirs 0.97 0.95 0.91

T o t ; 1 I 0 . 9 5 0.90

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cM = pt centimorpns per c11ron1osotne; cDNA =

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(41ro111oso111e corresponding to c1)N.A clones: genomics = loci per (.Iwot11oww cotwsponding t o single copy genomic clones; total =

loci col.l.esl)oll[litlg t o both cIINA and genomic clones; pachytene

= rel;ttive let~gtl~ of ptclytene cllrotnosomes (BARTON 19.50); mi- totic = relative length of mitotic (rnetapllase) chromosomes (LAPI-

TAN, G A N A I . ;tnd T A N K S I X Y 1989).

Inosomes and the high density areas occur near the end of the linkage groups. These two chromosomes are unique in that clusters of ribosomal genes are located adjacent to the centromeres

(5s

rDNA in chromosome I and 45s rDNA in chromosome 2). These ribosomal sequences have been localized both by RFLP mapping (Figure 1) and by in situ hybridi- zation (TANKSLEY et al. 1988, LAPITAN, GANAL and

1 ANKSLEY 199 1 ) and provide an approximate location of the centromere in the molecular linkage map for these chromosomes. In both instances the position of the Centromere coincides very closely to the areas of high marker density supporting the proposal that much of the clustering of markers on the linkage maps is around centromeres (Figure 3). For a few chromosomes ( 4 L , 7L, RS and

IIS),

areas of high marker density occur at the ends of the map and may correspond to telomeric areas which are reduced in recombination (Figure 1 and 3).

T o estimate the percentage of the total markers on

,..

the map located i n areas of high marker density we summed the number of markers in high density areas (defined as inter\& with >5 markers/2 cM) for each chromosome and divided by the number of total markers located on that chromosome. T h e values ranged from 1.5% for chromosomes 4 and 5 to 40% for chromosome I 2 with a mean of 28%. The areas of high density included markers corresponding to both cDNA clones and genomic clones in a relative frequency not significantly different from markers i n the rest of the genome.

T h e clustering of markers at centromeric and pos- sibly telomeric areas could be due to the interspecific nature of the cross used to construct the map. Inver- sions, or other chromosomal variations that differentiate species, are known to cause regional suppression of meiotic recombination (and hence clustering of markers on a linkage map) (BURNHAM 1962). How- ever, two lines of evidence argue against this expla- nation. First, cytological and genetic studies, using the same interspecific cross, have failed to detect any significant structural differences in the chromosomes ofthe two species (KHUSH and RICK 1963; RICK 1969). I n fact, all evidence thus far suggests that the chromosomes of L. esculentum and L. pennellii are con- served i n gene order and share homology in both single copy and repetitive sequences (ZAMIR and TANKSLEY 1988). Second, subsets of the molecular markers reported in this study have also been mapped i n other populations derived from crosses involving more closely related tomato species, as well as in populations derived from intraspecific crosses. While the overall levels of recombination may be higher in these more related crosses, the distribution pattern of crossing over is similar (PATERSON et al. 1988, 1991; M . W. (;ANAL, unpublished data). Similar observations have been made in potato when comparing maps made from different crosses (M. W. BONIERRALE, unpublished data).

Centromeric suppression of recombination: As-

suming a random distribution of markers, low levels of meiotic recombination would cause markers, that are physically well separated, to cluster on a linkage

map. Therefore, the higher density of markers i n centric regions may be an effect of lower levels of meiotic recombination. In Drosophila, up to a 40-fold suppression of recombination has been reported near the centromeres (ROBERTS 1965). There is evidence that two factors contribute to the suppression in Dro- sophila. First, the centromere is believed to exert a

direct, negative effect on crossing over in flanking chromosomal sequences, a phenomena termed the "centromere effect" or "spindle fiber effect" (BEADLE

(14)

Chromosome I

S. D. Tankslev

Chromosome 2

Chromosome 3 Chromosome 4

demonstrated in yeast where a cloned centromere from the third chromosome (CEN3) has been shown to decrease recombination when it is artificially inte-

grated into new sites in the genome (LAMRIE and KOEDER 1986). T h e second factor reducing recombination around centromeres in Drosophila is attrib- uted, not to the centromere, but to heterochromatin which is proximal to the centromeres (ROBERTS 1965). Heterochromatin experiences reduced levels of recombination compared with euchromatin, presumably due to the more condensed state of heterochromatin in meiosis at the time of crossing over (ROBERTS 1965).

Tomato chromosomes, like those of Drosophila, contain centromeric heterochromatin and it has been shown that, at least for some chromosomes, recombination is suppressed in the heterochromatic region around the centromeres and that this effect can be enhanced in wide crosses (KHUSH and RICK 1967, 1968; RICK 1969, 1972). T h e suppression of recombination in tomato may therefore result from both a centromeric effect and/or inherently lower levels of recombination in the heterochromatin around centromeres. T h e hypothesis that high density clustering of markers in centromeric regions is due to suppressed recombination, and not other factors, is supported by physical mapping data around one of the tomato

centromeres. GANAL, YOUNG and TANKSLEY (1989) constructed a partial restriction map, using pulsed field gel electrophoresis, around the Tm-2a gene (resistance to tobacco mosaic virus) which is known to be near the centromere on chromosonle 9. Results from that study indicate that 1 cM in this region of the map corresponds to more than 4 Mb-a value 6-fold greater than expected. In contrast, physical mapping around the 12 gene (conferring resistance to Fusarium race 3), which is located in euchromatin and distant from the centromere on chromosome 11, indicates a base pair to cM relationship more than IO-fold less than expected, suggesting much higher levels of recombination (SEGAL et al. 1992).