A molecular cytogenetic study of intergenomic recombination and introgression of chromosomal segments in lilies (Lilium)

Lilies (Lilium L.) are one of the most important ornamental bulbous crops for cut flower industry grown extensively in The Netherlands for last few decades. The genus Lilium consists of seven different sections with about 80 species. The species within genus Lilium (2n = 2x = 24) comprise a range of desirable and complementary characters. Besides being an important horticultural crop, lily (Lilium) also serves as an interesting model plant for molecular cytogenetic research and introgression breeding for several reasons like, i). Lily is a model crop for interspecific hybridization and it includes plants of different taxonomic species each of which possess valuable horticultural traits that need to be combined in the new cultivars. ii) Through careful selection n and 2n gametes can be obtained in interspecific hybrids. iii) The genomes of different species are so well differentiated genetically that the parental chromosomes can be clearly identified in the F1 hybrids as well as in the progenies through DNA in situ hybridization techniques. iv) The chromosomes are large enough and the number and position of homoeologous recombination sites can be clearly detected. v) The large and easily identified chromosomes in different lily species could be a potential source for the cytological mapping of the Lilium genomes. Taking advantage of these favourable attributes of lily, a molecular cytogenetic investigation was conducted to evaluate the amount of recombination and introgression of characters between Longiflorum - Asiatic (LA) and Oriental - Asiatic (OA) hybrids through the use of n and 2n gametes. For this purpose different F1 Longiflorum × Asiatic (LA) and Oriental × Asiatic (OA) hybrids were backcrossed with different Asiatic cultivars. Ovule and embryo rescue techniques were employed to get backcross (BC) progenies. Most of the F1 LA appeared to be sterile but some hybrids were able to produce only 2n gametes in considerable frequencies. However, in rare occasions it was also found that normal meiosis took place in few of the LA hybrids which resulted into the formation of normal n gametes. Ploidy level and intergenomic recombination was studied in LA interspecific hybrids in order to assess the possibility of functional n gametes and their potential use in introgression at diploid level in lily. A total of 104 BC1 LA interspecific lily hybrids were obtained, 27 diploids (2n = 2x = 24), 73 triploids (2n = 2x = 36) and 4 aneuploids (2x – 1, 2x + 2 or 2x + 3). Similarly, triploid BC1 (LAA) plants were backcrossed to diploid Asiatic parents. As a result 14 diploid BC2 progenies were produced. The intergenomic recombination and amount of introgression of respective genome (L and A) was assessed in these diploid genotypes through GISH (Genomic in situ Hybridization). Extensive intergenomic recombination was found among the chromosomes in LA hybrids. A large of amount of L- genome was transmitted from F1 LA hybrids to their subsequent BC1 progenies. However, very few segments of L- genome were introgressed from the BC1 diploid and triploid (LAA) plants to the BC2 progenies (Chapter 2). GISH identifies a considerable amount of recombination events amongst different interspecific lily hybrids (LA and OA) obtained from functional 2n gametes. Based on recombination sites on different chromosomes identified by GISH, cytological maps of three genomes of Lilium were constructed. For this purpose, BC progenies of two diploid interspecific hybrids of lily, viz., Longiflorum × Asiatic (LA) and Oriental × Asiatic (OA) were used. The BC progenies of LA hybrids consisted of both triploid (2n = 3x = 36) and diploid (2n = 2x = 24) with some aneuploid genotypes and those of OA hybrids mostly consisted of triploid (2n = 3x = 36) and some aneuploid genotypes. In LA hybrids 248 recombination sites were cytologically localized on 12 different chromosomes of each genomes (i.e., L and A). Similarly, 116 recombinant sites were marked on 12 chromosomes each from the BC progenies of OA hybrids (O and A genomes). The distances of the recombination sites from the centromeres are measured (in micrometres). Based on these recombination sites four cytological maps were constructed. Since an Asiatic parent was involved in both hybrids, viz., LA and OA, two maps were constructed for A genome which were indicated as Asiatic (L) and Asiatic (O) and one each for Longiflorum (A) and Oriental (A) genomes (Chapter 3). With a view to generate genetic variation via homoeologous recombination in BC progenies of LA and OA hybrids the most logical approach was the use of 2n gametes. 63 BC1 LA (LA × AA or AA × LA) and 53 OA (AA × OA) progeny plants were obtained after unilateral sexual polyploidization. 16 genotypes from F2 LA populations were obtained after bilateral sexual polyploidization through sib-mating of F1 LA hybrids. GISH was employed for the identification of the parental genomes, mode of origin of these progenies and measurement of the introgression in different interspecific lily hybrids. Most of the BC1 progeny plants (LA and OA) had originated through 2n gametes by First Division Restitution (FDR) mechanism. However, there were 12 genotypes in LA hybrids and four genotypes in OA hybrids that originated through 2n gametes formation as the result of Indeterminate Meiotic Restitution (IMR). A higher amount of recombination was found in LA hybrids as compared to OA hybrids. Intergenomic recombination was also determined in the sib-mated F2 LA population. In this case both parents had contributed gametes with the somatic number of chromosomes (i.e., 2n-2n) thus confirming the event of bilateral sexual polyploidization in interspecific LA hybrids. Based on these results, the relevance of interspecific lily hybrids obtained from uni- and bilateral sexual polyploidization leading to allotriploid and allotetraploid formation in interspecific lily hybrids is discussed in the context of introgression and mapping (Chapter 4). Molecular markers are an important tool for the construction of genetic linkage maps, as the first step in the genetic dissection of the required traits leading to crop improvement followed by the marker assisted breeding in different plants. Lilium has one of the largest genome in plant kingdom and genetic mapping in lilies is constrained by its large genome. DArT (Diversity Array Technology), a molecular marker technique can detect and type DNA variation at several hundred genomic loci in parallel without relying on genome sequence information. The DArT technique was developed for Longiflorum × Asiatic (LA) lily hybrids to enable an efficient and effective genetic mapping with the production of a large numbers of markers in microarrays-based assay. The restriction enzyme PstI + TaqI combination generated the largest frequency of polymorphic genomic representations for a genotyping array. Genomic representations from 88 F1 LA plants were used to assemble a DArT genotyping microarray. A total of 687 DArT markers were developed and 382 polymorphic markers were mapped on 14 main linkage groups which is two more then the haploid chromosome number (i.e. n = 12). The resulting linkage map with 382 DArT markers spanned 1329 cM (3.5 cM/marker on average). The results highlighted the potential of DArT as a genetic technique for genome profiling in the context of molecular breeding and genomics, especially in crops with large genome sizes where other techniques proved to be less useful (Chapter 5). The results of the present investigation are of practical implication. These results show the advantages of the n gametes and their subsequent progenies which opened a new approach of lily breeding ‘the analytic breeding’ in the allopolyploids. It also shows the possibility of using certain triploid hybrids for further breeding. A comparison has been made between different types of interspecific crosses, the amount of intergenomic recombination and introgressions of chromosomal segments obtained after unilateral sexual polyploidization. Furthermore, bilateral sexually polyploidization via sib-mated F1 hybrids producing 2n gametes has been proven. The use of allotetraploids obtained from bilateral sexual polyploidization could be a novel approach in the breeding of LA-hybrids. These allotetraploid with recombinant chromosomal segment may be a potential source to generate genetic variation in subsequent progenies. The molecular cytogenetic GISH and FISH techniques proved to be a powerful tool useful for the construction of cytogenetic maps in interspecific crosses in crops with large genomes sizes like lily. These techniques are also used for the identification and integration of genetic maps with chromosome maps. FISH also helps to monitor the introgressed chromosome segment or marker of interest in the subsequent progenies. Application of the DArT technique proved to be an effective method to construct genetic linkage maps especially crops (like Lilium) with large genome sizes where other techniques might be less useful.