A molecular cytogenetic analysis of chromosome behavior in Lilium hybrids

S.L. Xie

Research output: Thesisinternal PhD, WU

Abstract

 Lily (Lilium) has become one of the top bulbous crops for the cut flower industry in the past two decades. The genus Lilium comprises of approximately 80 species, which have been classified into seven sections. Each section possesses distinctive phenotypic characters, such as flower color, flower shape and  resistances to diseases and pests. Crosses between species in the same section are relatively easy and the resulting  hybrids are in general fertile, while interspecific crosses between species from different sections are rather difficult and the resulting  hybrids are in general sterile. As a result, different hybrid groups have been bred in the 20th century. Within these different hybrid groups, Longiflorum (L), Asiatic (A) and Oriental (O), which are derived from the section Leucolirion, Sinomartagon and Archelirion respectively, are of  commercial importance and hence, are the most widely cultivated lilies worldwide.

Lily hybrids provide an ideal model for molecular cytogenetic research. With the development of techniques of overcoming pre- and post- crossing barriers of interspecific crosses, as well as the application of asexual and sexual polyploidization to restore the fertility of F1 lily hybrids, combining of  desirable traits from different hybrid groups has become feasible. As a result, interspecific hybridization and polyploidization have been widely used in the breeding of new cultivars of lily. These cultivars, as well as other breeding materials from interspecific crosses, facilitate the application of molecular cytogenetic analysis due to three reasons: 1) the chromosomes of lily are large enough for cytological observations; 2) genomes of different hybrid groups are homoeologous; and 3) these homoeologous genomes can be simultaneously distinguished by DNA in situ hybridization. Using these lily hybrids combined with genomic in situ hybridization (GISH) and florescence in situ hybridization (FISH), the interaction of homoeologous genomes can be studied though meiotic observation of the F1 hybrids. Meanwhile, chromosome sequential variation with relevance to crossover and chromosome rearrangements can also be observed.

For this purpose, interspecific crosses between the Lilium longiflorum cultivar ‘White Fox’ and the Asiatic cultivar ‘Connecticut King’ were made, and some of these F1 hybrids, which show a relatively high fertility with the production of unreduced gametes, were backcrossed with an Asiatic cultivar . The meiosis of the interspecific hybrids, as well as the sexual polyploidized progenies, were analysed by GISH and FISH. In addition, one population of sexual polyploidized AOA hybrids was also analysed for the genome composition. Results showed that there was no evidence that lily allopolyploids possess any noticeable chromosome rearrangements. The equal segregation of reciprocal and non-reciprocal recombinant product showed that the intergenomic recombination in the sexual polyploidized progenies was indeed from a natural process-chiasmata formation and crossovers and hence, should not be considered as translocations as was suggested in literature for intergenomic recombination. This conclusion was further confirmed by meiotic observation of the interspecific F1 hybrids.

Detailed meiotic observations were carried out in interspecific hybrids between Longiflorum × Asiatic groups of lilies (Lilium) which were  used as parents to generate sexual polyploids with intergenomic recombination. Bivalents involving two homoeologous chromosomes, as well as unpaired univalents were the main configurations at metaphase I. However, in two genotypes, multivalents and bivalents both involving non-homologous pairing of two Asiatic chromosomes were observed. This indicated the presence of a duplication which was common to two non-homologous chromosomes in the hybrids. It is deduced that there was a reciprocal translocation in the Asiatic parent cv. ‘Connecticut King’ and these two genotypes resulted from duplication-deficiency gametes. Results from Anaphase I showed that chiasma formation involving non-sister chromatids gave rise to two strand single, two strand double, three strand double, four strand double and multiple exchanges. It is also noticeable that there was a high frequency of multiple crossovers in the genotypes with duplication, indicating a reduced crossover interference in multivalents. Beside the normal crossovers, also chromosome bridges at anaphase I of meiosis were  observed. GISH and FISH painting showed that these bridges involve not only non-sister chromatids but also sister-chromatids. The bridges, without any differentiation along their length, were always accompanied by fragments with a variable size. These results indicated that the bridges, together with the accompanying fragments, were derived from U-type exchanges. Other than homologous recombination (HR), nonhomologous end joining (NHEJ) probably led to the production of bridges when repairing the double strand breaks (DSBs) during meiosis.

Progenies from unilateral polyploidization of crosses between LA hybrids and Asiatic cultivars  were predominant triploids. However, three exceptional plants, which possessed 35 normal chromosomes and a small aberrant chromosome instead of the expected normal number of 36, were observed. In all three cases the small aberrant chromosomes were isochromosomes which had obviously originated during the first backcross generation, and the length of the arms of these aberrant chromosomes were always related with the length of the short arm of the missing chromosome. Furthermore, one of these three chromosomes possessed 45S rDNA hybridization sites in the proximal positions, which resembles the short arm of the missing chromosome (chromosome 4 of L genome). Combined with the results of chromosome breakage during meiosis, centric breakage and fusion is a putative mechanism of the production of these isochromosomes. Meanwhile, two small, supernumerary or B chromosomes were detected as extra chromosomes in a tetraploid plant derived from chromosome doubling of an intersectional hybrid (2n=2x=24) between a cultivar of the Longiflorum (L) and the Trumpet (T)  group. When this tetraploid LLTT hybrid was crossed with a triploid LLO hybrid (O=Oriental), the B chromosome was transmitted to 73.4% of the progenies. Based on GISH and FISH characterization it was shown that the B chromosome found consisted of two identical arms, with 5S rDNA hybridizing to the majority of it, which were flanked by normal telomeres, suggesting that this is an isochromosome.

The results of current investigations are of practical implication for a number of reasons. Firstly, the behavior of homoeologous chromosomes during meiotic processes in lily hybrids was  studied in detail, and it can be used to explain the profound genetic changes in the early generations during hybrid speciation. Secondly, some problems that go unnoticed in genetic mapping can be predicted and well explained by the occurrence of chromosome rearrangements in the parents which are used to produce the segregation population and  thirdly, the discovery of U-type exchanges during meiosis and de novo isochromosomes in the backcross progenies supplies an alternative mechanism for the origin of B chromosomes.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • Visser, Richard, Promotor
  • van Tuyl, Jaap, Promotor
  • Arens, Paul, Co-promotor
Award date6 Jun 2012
Place of PublicationS.l.
Print ISBNs9789461732248
Publication statusPublished - 2012

Keywords

  • lilium
  • cytogenetics
  • molecular genetics
  • genetic analysis
  • hybrids
  • chromosomes
  • recombination
  • interspecific hybridization
  • meiosis

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