Introgression of tomato chromosomes into the potato genome : an analysis through molecular marker and in situ hybridisation techniques = [Introgressie van tomatenchromosomen in het aardappelgenoom : een analyse met behulp van moleculaire merker en in situ hybridisatie technieken]

Transfer of alien chromosomes and genes across intergeneric boundaries can be useful not only for the introgression of desirable characters but also for fundamental genetic studies. The successful demonstration of hybridisation of potato ( Solanum tuberosum ) and tomato ( Lycopersicon esculentum ) through protoplast fusion in 1978, created the potential for introgressing chromosomes and genes from one genus into the other. However, real prospects of adding tomato chromosomes into the potato genome arose only after a potato (+) tomato fusion hybrid was successfully backcrossed to tetraploid potato in 1994.

Despite this achievement, the problem of selective alien tomato chromosome elimination from the fusion hybrid and the backcross progenies, as well as the behaviour of the alien tomato chromosomes in the potato background, raised the following questions: (i) in view of the selective elimination of alien tomato chromosomes, is it possible to establish a complete series of alien tomato chromosome additions in a potato background? (ii) do the introgressed alien chromosomes retain their structural integrity? (iii) can all the alien tomato chromosomes be transmitted through the gametes to the sexual progeny and if so, in what frequency? (iv) if there are any structural changes in the introgressed alien chromosomes, can they be identified and characterised? (v) do the potato and tomato chromosomes pair homoeologously and cross-over?

An attempt was made in the present investigation to create the appropriate plant material to answer the five above mentioned questions. In this connection, the chromosome constitution of the somatic fusion hybrids and their backcross derivatives, viz., BC ₁ , BC ₂ and BC ₃ progenies were analysed through a combination of restriction fragment length polymorphism (RFLP) using tomato chromosome specific probes, genomic in situ hybridisation (GISH) and fluorescence in situ hybridisation (FISH) analyses.

In order to produce BC ₁ progenies with the full potential for the creation of all the 12 possible monosomic tomato chromosome additions in a tetraploid potato background, three different hexaploid somatic fusion hybrid genotypes, viz., C31-17-5, C31-17-24, and C31-17-51, were used successfully as female parents in crosses with four tetraploid potato clones; cv Katahdin, cv Frieslander, 6704-1 and AM 66.42 (chapter 2). From these crosses, several BC ₁ progenies were obtained. Among these, six BC ₁ plants, viz., 6739, 2001, 2002, 2003, 2004 and 2005 were analysed through RFLP and GISH analyses. Due to preferential alien tomato chromosome elimination, a variable number of tomato chromosomes (6-11) was present in these genotypes. An important result was that all the 12 different tomato chromosomes were present in these six BC ₁ progeny plants, albeit in different genotypes.

By backcrossing three of these BC ₁ plants to tetraploid potato, a total of 97 BC ₂ plants were obtained and analysed through RFLP and GISH (chapter 3). The number of alien tomato chromosomes in these BC ₂ plants varied from 0-6. The average rate of tomato chromosome transmission among different populations ranged from 1.7 to 3.4. Generally, a much higher rate of transmission was observed when the alien tomato chromosome in the BC ₁ plant was in a disomic condition. A statistical analysis of the rate of female transmission could not prove an equal probability of chromosome transmission among the individual tomato chromosomes. A notable result of this investigation on the BC ₂ plants was that among the 12 possible types of monosomic additions seven, viz., chromosome 1, 2, 4, 6, 8, 10 and 12, were identified. This clearly indicated that, despite the problem of selective elimination of the alien tomato chromosomes, it is possible to select genotypes that retain the alien chromosomes in a stable condition and transmit them through the gametes to the progeny.

Besides monosomic additions, an occasional disomic was also detected through RFLP and GISH analyses (chapter 4). Remarkably, one of the chromosomes of this pair in the BC ₂ plant 2101-1, was much smaller that the other. A detailed analysis of microsporogenesis indicated that at pachytene stage the large and small tomato chromosomes paired as homologues, and morphologically it was identified as chromosome 10 of tomato, which was also concurrent with RFLP analysis using chromosome specific probes. Morphologically, a deletion for the heterochromatic part of the long arm of the chromosome 10 of tomato had given rise to the smaller of this homologous pair. A detailed analysis of microsporogenesis indicated that a precocious disjunction and division of the alien pair of chromosomes was the cause of the origin of disomic additions in this material. It was evident from this study that the pachytene chromosomes of tomato can be clearly identified after GISH procedures even in a polyploid genotype and the deletion could be clearly characterised. In addition, a BC ₃ population consisting of 96 plants was created and characterised through RFLP and GISH analyses after backcrossing the disomic addition to a tetraploid potato pollen parent. Therefore, the female transmission of both the normal and the aberrant homologues could be determined.

To gain insight into homoeologous pairing and crossing-over between the potato and tomato chromosomes, a somatic fusion hybrid, C31-17-51, with two translocated chromosomes, and two genotypes, 2103-1 and 2301-2, with monosomic alien tomato additions for chromosome 1 and 8 respectively, were investigated (chapter 5). There was clear evidence for the presence of a reciprocal translocation in the somatic fusion hybrid. As a consequence, the quadrivalent formation involving potato and tomato chromosomes was only due to the autosyndetic pairing. In the absence of any knowledge of this translocation, the high degree of pairing, 86.0 %, could have been mistaken for allosyndetic pairing. An analysis of chromosome pairing and chiasmata formation in the monosomic additions indicated a very low frequency of homoeologous pairing, 1.3 and 1.1 % for the chromosome additions 1 and 8 respectively. Chiasma formation between homoeologues chromosomes was estimated to occur in 0.8 % of the meiotic cells studied in the monosomic alien addition for chromosome 8.

The investigations presented in this thesis demonstrated that, although the frequency of homoeologous pairing is low, there is a prospect for introgressing tomato chromosomal DNA into the potato genome.