IDENTIFICATION OF PARENTAL CHROMOSOMES AND DETECTION OF RIBOSOMAL DNA SEQUENCES IN INTERSPECIFIC HYBRIDS OF LILIUM REVEALED BY MULTICOLOR IN SITU HYBRIDIZATION

Abstract

In wide interspecific crosses of lily, different hybrid groups were used viz., L. longiflorum (L), Asiatic hybrids (A) and Oriental hybrid (O). Two BC1 plants, OLA and OLO, were derived from crossing Oriental hybrids as a mother to LA and LOLO hybrids as male parent. LA and LOLO F1 hybrids were known to produce 2n-gametes ( 40%), respectively. GISH enabled unequivocally the identification of parental chromosomes. Both BC1 progeny, OLA and OLO, possessed three genome sets with 36 chromosomes (2n=3x=36) without any intergenomic recombinations. 45S rDNA probe was successfully hybridized on NOR bearing chromosomes of L. longiflorum and Oriental hybrid. L. longiflorum possessed 3 rDNA signals on chromosome 3, 4 and 7. Chromosome 3 of L. longiflorum showed one signal near to the secondary constriction on the long arm. 9 chromosomes of Oriental hybrid showed one or two hybridization signals indicating some extent of polymorphism. This polymorphism confirmed the fact that Oriental hybrids are mixed with several species in the section Archelirion. Based on result of the polymorphism of the rDNA signal, the origin of some chromosome number of Oriental hybrid could be assumed. INTRODUCTION Lilium, a large genus with over 80 species, is classified into seven taxonomic sections (Comber 1947). Because of its large genome size (L. longiflorum = 141.1 pg/cell, Bennett and Smith 1976), the genus Lilium has furnished material for cytological research since Strasburger (1880). All species are endemic to Asia, North America and Europe of the Northern Hemisphere. Identification of recombinant chromosome or chromosome segments in interspecific hybrid progenies would be beneficial for introgression breeding. Furthermore, it is important to identify the exact chromosome number and breakpoint of the recombinant chromosomes for the probable introgressed genes and localization of gene(s) on the chromosome along with phenotypic characters. In order to identify the parental chromosomes of the interspecific hybrid, genomic in situ hybridization (GISH) is the most powerful tool. Physical localization of rDNA genes was used as a useful marker to distinguish between NOR containing chromosomes and provides valuable evidence about the genome evolution at molecular cytogenetic point of view (Gerlach and Dyer 1980; Kamstra et al., 1997). 5S and 18S-25S rDNA probes corresponding to the site of ribosomal RNA genes were detected through fluorescence in situ hybridization in many crops (Mukai et al., 1991). Species specific sequence together with FISH is also useful markers to identify the rest of chromosomes, which can not be distinguished through banding techniques (Kamstra et al., 1997). The aim of the present study was identification of individual chromosomes of L. Proc. 8th Int. Symp. on Flowerbulbs Eds. G. Littlejohn et al. Acta Hort. 570, ISHS 2002 404 longiflorum and Oriental hybrid by means of GISH and FISH. MATERIAL AND METHODS Plant Material OLA-hybrid ‘942653-1’ was derived from Oriental hybrid ‘San Marco’ (O) × LA-hybrid ‘88542-69’. F1 LA-hybrid was known to produce fertile 2n-pollen by FDR about 9.6 % and was used as male parent for BC plant production. Since the LO-hybrid was sterile, an amphidiploid ‘950232’ (LOLO) was made by doubling the chromosome number of the LO-hybrid. The triploid ‘972729-4’(2n=3x=36; OLO), the interspecific hybrid of Oriental hybrid ‘Mero Star’ (O) × LO-hybrid, was used for GISH and FISH analysis. Interspecific hybrids were produced through integrated pollination and embryo rescue methods (Van Tuyl et al.,. 1991). Plants were grown in a greenhouse at 20 – 25°C during the day and 14 – 18°C during the night. Chromosome Preparation For the study of mitotic metaphase complements, the fast growing root tips were collected in the morning and pretreated in saturated α-bromonaphtalene solution for 2 hours at 20 °C followed by an overnight treatment at 4 °C. The material was fixed in Carnoy’s solution (acetic acid:ethanol = 1:3) and stored at –20 °C until use. Before making squash preparations, root tips was incubated in a pectolytic enzyme mixture containing 0.3% pectolyase Y23, 0.3% cellulase RS and 0.3% cytohelicase in 10mM citrate buffer (pH 4.5) for about 1 – 1.5 hour at 37 °C. Squash preparations were made in 60% acetic acid. Slides were frozen in liquid nitrogen and the cover slips were removed by using a razor blade. Slides were finally dehydrated in absolute ethanol for a few minutes, dried and stored at –20 °C until use. DNA Probes Preparation Total genomic DNA of L. longiflorum ‘Snow Queen’ was used as a probe. The probe DNA was labeled with digoxigenin-11-dUTP by nick translation according to the manufacturer’s instructions (Boehringer Mannheim). Clone pTa71 contains the 9 kb EcoRI fragment of 45 S ribosomal DNA from wheat (Gerlach and Bedbrook 1979). Isolated DNA of 45 S rDNA sequences from pTa71 was labeled with either biotin-16dUTP by nick translation for in situ hybridisation according to the manufacturer’s manual (Boehringer Mannheim, Germany). Blocking DNA was obtained by autoclaving herring sperm DNA for 5 min at 121°C. The size of blocking DNA ranged from 100 to 500 bp. Because of Oriental hybrids are derived from at least 3 species of Archelirion section, 45S ribosomal DNA sequences were hybridized to the mitotic chromosome complement of OLO-hybrid. Fluorescence in Situ Hybridization (FISH) The in situ hybridization protocol was carried out according to Kuipers et al., (1997) with minor modifications. Briefly, slides were pretreated with RNase A (100 μg/mL) for 1 hour and pepsin (5 μg/mL) for 10 min, both at 37°C, followed by formaldehyde (4%) for 10 min at 20°C, dehydration with 70%, 90% and absolute ethanol for 3 min and air dried. Hybridization followed using a mixture consisting of 2x SSC, 50% formamide, 10% sodium dextran sulfate, 0.25% SDS, 2.0 ng/μL digoxigenin-11dUTP labeled total genomic DNA of L. longiflorum ‘Snow Queen’ and 30-40 ng/μL herring sperm DNA for blocking. DNA was denatured by heating the hybridization mixture at 70°C for 10 min and then placed on ice for 10 min. For each slide 40 μL hybridization mixture was used. The preparations were denatured at 80°C for 10 min. After overnight hybridization at 37°C in humid chamber, slides were washed at room temperature in 2x SSC for 15 min, 0.1x SSC at 42°C for 30 min. The digoxigenin labeled probe DNA was detected with 20 μg/mL anti-dig-FITC (fluorescein isothiocyanate; Boehringer Mannheim) and 20 μg/mL rabbit-anti-sheep-FITC (Vector Laboratory).