Theor Appl Genet (1992) 83:1027 1034

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9 Springer-Verlag 1992

Lycopersicon esculentum lines containing small overlapping introgressions from L. pennellii Y. Eshed, M. Abu-Abied, Y. Saranga, and D. Zamir * Faculty of Agriculture, The Hebrew University of Jerusalem, Department of Field and Vegetable Crops, and the Otto Warburg Center for Biotechnology, 76100 Rehovot, Israel Received June 27, 1991; Accepted October 1, 1991 Communicated by F. Salamini

Summary. The objective of this project was to introgress small overlapping chromosome segments which cover the genome of L. pennellii into Lycopersieon esculentum lines. The interspecific hybrid was backcrossed to L. esculentum, and a map of 981 cM, based on 146 molecular markers covering the entire genome, was produced. A similar backcross 1 population was selfed for six generations, under strong selection for cultivated tomato phenotypes, to produce 120 introgression lines. The introgression lines were assayed for the above-mentioned molecular markers, and 21 lines covering 936 cM of L. pennellii, with an average introgression of 86 cM, were selected to provide a resource for the mapping of new D N A clones. The rest of the lines have shorter introgressions consisting of specific regions with an average size of 38 cM. The proportion of the L. pennellii genome in the introgression lines was lower than expected (252 cM) because of strong selection against the wild-parent phenotype. The mean introgression rate for ends of linkage groups in the 120 lines was 3 times higher than for other regions of the genome. The introgression lines can assist in RFLP-based gene cloning by allowing the rapid selection of D N A markers that map to specific chromosome segments. The introgression lines also provide a base population for the mapping and breeding for quantitative traits such as salt and drought tolerance that characterize the wild species L. pennellii.

Key words: Lycopersicon esculentum - L. pennellii RFLP - Introgression lines - Breeding

* To whom correspondence should be addressed: Faculty of Agriculture, The Hebrew University of Jerusalem, P.O. Box 12, 76100 Rehovot, Israel

Introduction Restriction fragment length polymorphisms (RFLPs) provide a system for high resolution mapping of genomes. In plants, various populations have been subjected to RFLP mapping analysis. (1) F2 and backcross (BC) generations; these traditional mapping populations were produced either within or between species, depending on the level of RFLPs in the analyzed genomes (Tanksley et al. 1989). (2) Recombinant inbreds (RI); such lines have been developed in maize, which is a highly polymorphic species, as a result of the inbreeding of F2 populations (Burr et al. 1988). The main advantage of RI over the F 2 and BC populations is that it constitutes a permanent mapping resource that can be propagated from seed and analyzed by a number of investigators whose results are cumulative. RI lines can be used for the analysis of quantitative traits since each inbred genotype can be planted in replicated trials (Burr and Burr 1991). (3) Cytogenetic variants; uneuploid stocks allow a rapid assignment of D N A clones to chromosomes through a comparison of the relative intensities of the bands produced in Southern blots (Young et al. 1987; Sharp et al. 1989). In maize, RFLP loci can be mapped to chromosome regions using B - A translocations (Weber and Helentjaris 1989). (4) Nearly isogenic lines (NILs); lines differing in only a small chromosome segment were used to map D N A clones that are tightly linked to disease resistance genes in tomato (Young et al. 1988; Sarfatti et al. 1989; Levesque et al. 1990; Messeguer et al. 1991; Klein-Lankhorst et al. 1991 a; Martin et al. 1991; Sarfatti et al. 1991; Behare et al. 1991). Such clones can be identified because resistance genes have been introgressed into cultivated varieties from wild relatives and the process of introgression has left a residual chromosome segment of polymorphic D N A flanking the gene of interest.

1028 The introgression lines described in this study provide a set of N I L s for a large p o r t i o n of the t o m a t o genome. Lycopersicon pennellii is a green-fruited species commonly used as the material of choice for genetic studies in tomato. The accession LA716, which was collected in the Peruvian desert, is self-compatible, highly inbred and can be easily hybridized to L. esculentum (Rick and Tanksley 1981). The t o m a t o R F L P map, which includes more than 600 markers covering the 12 chromosomes, was developed through analysis o f an F 2 p o p u l a t i o n resulting from a cross between the cultivated t o m a t o L. esculentum and L. pennellii (Tanksley et al. 1989). The interspecific p o p u l a t i o n was used for mapping, since the degree of p o l y m o r p h i s m detected by R F L P analysis between L. esculentum and L. pennellii is more than 15 times higher than between L. esculentum accessions (Miller and Tanksley 1990). In this study we used the t o m a t o R F L P system to construct a p o p u l a t i o n of plants containing short overlapping introgressions o f the L. pennellii genome in a processing t o m a t o genetic background.

Materials and methods

BC1 mapping population For mapping analysis L. esculentum cv 'Vendor' was crossed as the female parent to L. pennellii (LA7t6). The interspecific hybrid was backcrossed as the male parent to 'Vendor', and 119 BC1 progenies were grown and subjected to RFLP analysis.

Introgression lines The L. eseulentum processing tomato cv. 'M82-1-8' was crossed to L. pennellii (LA716), and the F 1 hybrid backcrossed to 'M821-8'. Of the 600 BC1 plants grown in the field in 1984, 99 were selected on the basis of seed availability. During the following year 12 individuals from each of the 99 selfed BCt plants (BCISt) were grown and selected for L. esculentum characteristics such as growth habit, fruit color, fruit size and yield. This basic scheme of selfing and single plant selections in the field (an average of 1500 plants grown annually, of which approximately 100 were selected for the next generation) was continued until BCI $6, at which stage 120 plants representing 20 of the original BC1 individuals were chosen for RFLP analysis. Field selection was conducted in the Negev Desert, Israel, where plants were drip-irrigated with saline water of 10-20 dS/m according to Saranga et al. (1991).

RFLP analysis DNA was extracted from 'Vendor', 'M82-1-8', L. pennellii, the interspecific hybrids, the BCI mapping population and the BC1S6 plants. DNA isolation, restriction digests, electrophoresis on agarose gels, Southern blots, hybridizations and autoradiography were as described by Bernatzky and Tanksley (1986), except that filters were probed with random hexamer-labeled plasmids (Feinberg and Vogelstein 1983). In order to determine the RFLPs for analysis of the BC1 and BC 1S6 populations, 134 random genomic and cDNA clones (of which 5 identified unlinked duplicated loci) covering the entire genome were hybridized to survey filters carrying DNA of

L. esculentum ('Vendor' and 'M82-1-8'), L. pennellii (LA716) and the interspecific hybrids digested with the following restriction enzymes: EcoRI, EcoRV, DraI, HaeIII, XbaI and BstNI. Clones were hybridized individually to the populations on the basis of the appropriate polymorphism between the parents. Markers that cosegregated in the BCI population were not assayed in the BCIS6 (Table 1). Isozyme analysis for the loci Prx-1, Pgm-2, Aps-1, Aps-2, Got-2, 6Pgdh-2 and Pgi-1 was performed according to Zamir and Tal (1987).

Mapping analysis Linkage maps were constructed using MapMaker (Lander et al. 1987) and employing the Kosambi mapping function. Markers were ordered along the linkage groups only if they exhibited preferred orientation with a Lod score greater than two. The order was verified by manual examination of the data in threepoint linkage tests for minimum number of double recombinants.

Genome composition Genome composition in the BC1 and introgression lines was estimated according to Paterson et al. (t 991). When consecutive markers along the chromosomes of an individual showed the same genotype, it was assumed that the region intervening between the markers was composed entirely of the marker genotype. When consecutive markers along the chromosomes of an individual showed different genotypes, it was assumed that half of each genotype made up the whole interval. Such estimates disregard the possibility of double recombinants within an interval. To compare the BCI population and the introgression lines, we calculated genome composition on the basis of a haploid genome. This approach yields the same composition estimate for an introgression line homozygous to an L. pennellii segment as it does for an introgression lines heterozygous to the same segment. The expected total length (ETL) of chromosome segments carrying the L. pennellii alleles, either in the homozygous or heterozygous state, out of the total map length (TML) after n generations of selfing of a BCI population is: ETL = {[0.5n+ (0.5-0.5~)/2] x TML}. The expected ratio (ER) of heterozygous introgressed segments out of the total introgressed segments after n generations of selfing of a BC1 population is: ER = {(0.5n)/[0.5n-l-(0.5--0.5n)/2]}.

Results

The B C I mapping analysis The R F L P map, based on 119 BC1 individuals, includes 146 molecular markers (Fig. 1). The restriction enzymes used for m a p p i n g and the number o f BCI plants scored are shown in Table 1. Total m a p length was 981 c M and the average distance between the markers was 8.4 cM. The mean number o f recombination events for a BC1 individual over its entire genome was 8.8, with a minim u m of 3 and a m a x i m u m of 17. A n average diploid BCI individual would be expected to contain 50% o f the L. pennellii genome (490 cM) in a heterozygous state. In our population an average BC1 plant contained 519 c M from L. pennellii with a minim u m o f 237 cM and a m a x i m u m o f 794 cM (Fig. 2). The

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Fig, 1. RFLP linkage map of the tomato developed by segregation analysis in a BC1 generation derived from an L. esculentum x L. pennellii cross. Markers at the bottom of the chromosomes cosegregated with the markers having the same numbers in brackets. The black regions represent the portion of the genome included in the introgression lines

higher than expected inclusion of L. pennellii chromosome segments in the BC1 resulted from deviations from the expected Mendelian ratios. Regions on chromosomes i, 2, 6, 9, 10, 11 and 12 showed a significant excess of L. pennellii alleles, and the L. esculentum alleles were favored only for chromosome 8 (Table 1). Introgression lines From the selected BCIS6 lines, 120 plants were scored for 129 molecular markers. With no selection, an average

BC1S6 plant would be expected to contain 252..5 cM of the wild parent, of which 88.5% would be in a homozygous state. The genome composition of the BC1S6 population indicates that the mean inclusion of the wildparent genome was 47 cM (Fig. 2), of which 68% were in a homozygous condition. The mean number of independent introgressions for an average plant in the population was 2.61. Four regions on chromosomes 3, 6, 8 and 12 were selected out during the breeding of the introgression lines (Fig. 1). The data indicate that, in general, the chromo-

1030 Table 1. Segregation of 129 markers in a BCI generation derived from an L. esculentum x L. pennellii hybrid and the number of BC1S6 introgression lines (ILs) containing the L. pennellii allele at each locus

Table 1. cont.

Locus

Mapping enzyme a

EE : EP b

Number of ILs with L, pennellii allele

Chromosome 1 Prx-1 TG24 2 TG334 5 TG224 2 Sod-2 3 TG59 4 TG71 4 TG335 5 TG245 5 TG430 3 TG88 3 TG85 4 TG53 4 TGI58 5 TG159 5 TG259 6

54:53 54 : 52 43:45 43 : 45 52:61 59: 54 56: 56 33 : 55 * 31 : 58 ** 27 : 41 36:46 41 : 56 47: 58 37:51 40 : 49 51 : 56

2/120 (1) c 1/119 1/120 2/120 3/119 3/115 2/120 2/120 7/120 7/120 8/120 8/120 7/116 10/120 8/120 8/120

Chromosome 2 TG31 3 TG1B 1 TG165 3 TG14 1 TG308 2 TG332 5 TM6 5 TG131B 3 TG48 2 TG34 6 TG204 4

44:65 45 : 67 * 20 : 31 48 : 66 33:43 36 : 49 43:49 48:68 50: 61 52 : 65 47 : 56

7/120 (5) 8/120 8/120 8/116 12/120 13/120 12/120 12/120 4/120 4/119 8/108 (4)

Chromosome 3 TG40 2 TG135 6 TG130 4 TG66 1 TG39 2 TG134 1 TG284A 3 TG126 4 TG244 5 TG89 4 TG94 4

46:48 61:55 63:56 59:60 56 : 58 54: 54 38 : 47 40:45 54:44 63 : 54 23:29

18/120 (7) 11/120 2/120 0/120 0/120 0/120 1/120 1/120 3/120 (2) 1/117 2/120 (1)

Chromosome 4 TGI 5 2 TG123 3 TG182 2 Pgm-2 TG208 2 TG284B 3 TG62 2 TG65 4 TG305 1 TG374 2 TG22 3 TG37 5

49 : 50 59:58 23:36 54: 59 41 : 38 46 : 31 49:44 46 : 55 39:28 32 : 25 47 : 42 56:37

4/116 9/120 9/118 2/120 2/120 3/116 2/119 2/120 6/118 (1) 5/120 6/120 6/120

Locus

Mapping enzyme a

EE : EP b

Number of ILs with L. pennellii allele

Chromosome 5 TG441 4 CD41 4 CD 31 6 TG96 4 TM5 1 TG100 2 TG318 2 TG358 3 TG23 1 TG351 3 TG60 3 TG69 3 TG185 4

33:33 45: 52 54: 49 56: 56 53 : 54 58: 58 57: 59 41:26 56: 63 36:36 53 : 61 44: 53 42:54

3/120 4/120 2/120 2/120 2/120 4/120 2/120 3/120 9/119 8/120 11/120 13/120 16/119

Chromosome 6 TG231 4 Aps-1 TG25 1 TG54 3 TG253 3 TG275 1 TG115 3 TG258 3

26:48* 53:65 44:51 44: 59 47:53 54: 61 58 : 60 47:52

4/119 4/120 1/118 2/120 2/120 1/120 5/120 7/120 (2)

Chromosome 7 TG199 l TG20 2 TG217 6 Got-2 TG183 4 TG143 2 TG166 3 TGI3 3 TG272 2

50:68 49 : 61 51:68 48 : 53 54:64 53:66 52 : 67 52:67 37: 49

5/111 (2) 3/120 4/120 7/120 16/117 16/117 3/120 3/120 1/120

Chromosome 8 TG294 2 PNP24 3 TG330 2 Aps-2 TG16 4 TGl17 6 TGI76 3

54:40 64: 43 54:37 72 : 46 * 72:45** 63:47* 51:41

11/120 (5) 6/116 5/120 0/120 0/120 2/120 (1) 1/119

32:53* 42:63 * 41:71 ** 38 : 66"* 49 : 59 36:53 52:63 59 : 60 60 : 58 39 : 43 33:40 57 : 53 44 : 44

7/119 (1) 6/120 7/105 7/108 11/120 15/120 13/119 1/120 3/120 i/119 1/120 1/120 7/120 (6)

(1) (2)

(1) (1)

(7)

Chromosome 9 TG254 TG105A TG18 TG9 P3 TG225 TGI0 TG79 TG317 TG248 TG421 TG8 TG328

1 3 1 1 2 2 1 4 1 2 3 3 5

1031 Table 1. cont. Locus

Mapping enzyme ~

EE: EP b

Number of ILs with L. pennellii allele

Line

Chromosome 10 TG313 1 TG303 4 TG43 2 TGIA 1 TG52 1 TGI03 4 TG285 3 TG420 1 TG229 1 TG63 3

33:34 32:33 28:43 54:65 53:63 56:55 27:47* 24 : 38 22:70*** 31 : 67 ***

5/119 6/120 3/117 5/119 5/119 6/118 6/120 3/119 15/117 (3) 22/120 (10)

Chromosome 11 TG194 3 TG108 2 Sod-1 2 TG36 1 TG30 4 TGI05B 3 TG26 4 TGI04 2

33:46 37:81 *** 38:75*** 31:73 *** 24:63*** 40:77 *** 48:70* 22:33

29/119 (11) 18/119 9/110 12/120 11/119 11/120 11/119 21/117 (10)

Chromosome 12 TG473 2 TG296 2 TG28A 1 6Pgdh-2 TG124 4 Pgi-I TG28B 1 TG263 3 TG68 2 TG149 3

37:30 32:44 44:61 37 : 8t *** 30:75 *** 41:78 *** 43:74*** 23:43 ** 55 : 63 48 : 50

10/119 (10) 1/120 1/120 0/120 0/120 0/120 0/120 4/120 4/120 23/103 (19)

a 1, EcoRI; 2, EcoRV; 3, DraI; 4, HaelII; 5, BstNI; 6 XbaI u EE, Homozygous to the L. esculentum allele; EP, heterozygous to the L. pennellii allele. Chi-square values greater than this would be expected by chance at a probability (P): * 0.01