Genome Analysis of Lilium tigrinum by Chromosome Microdissection and Molecular Cytogenetic Techniques (original) (raw)
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Euphytica, 2022
This study was aimed at differentiating parental genomes, examining intergenomic composition, and mapping mitotic metaphase chromosomes by localizing parental and 18S rDNA probes in seven interspeci c hybrid progenies that originated from Lilium longi orum. Since in situ hybridization has not been previously used in lily breeding, ow cytometry was used in conjunction with genomic and uorescent in situ hybridization to determine the genomic contribution of each parent to the interspeci c progenies. A signi cant variation was observed in the DNA content, chromosome length, and 18S loci in F 1 as compared to the female and male parents. L. longi orum showed nearly two times higher DNA content than the male parents and L. longi orum × Asiatic progenies, but eight times higher than L. longi orum × L. hansonii. Genomic in situ hybridization results revealed that both female and male parents contributed an equal number of chromosomes to their interspeci c F 1 offspring. Fluorescent in situ hybridization mapping revealed that 18S rDNA had 8, 6 and 7 loci in L. longi orum parents, i.e., White heaven, Bright tower, and White tower, respectively, whereas each Asiatic cultivar and L. hansonii used as male showed 8 and 12 loci respectively. Interspeci c progenies showed 8 and 7 loci in LA, and 10-11 in LM hybrids. These cytogenetic results implied equal genetic and chromosomal contribution from both parents to their intergenomic progenies. Therefore, this combined (Schwarzacher et al., 1992)cytogenetic method has the potential to be an affordable and time-saving approach in lily breeding that could determine the status of hybrids and their genomic origin while achieving physical mapping and detecting genes in different genomes.
Genome, 2001
Detailed karyotypes of Lilium longiflorum and L. rubellum were constructed on the basis of chromosome arm lengths, C-banding, AgNO 3 staining, and PI-DAPI banding, together with fluorescence in situ hybridisation (FISH) with the 5S and 45S rDNA sequences as probes. The C-banding patterns that were obtained with the standard BSG technique revealed only few minor bands on heterologous positions of the L. longiflorum and L. rubellum chromosomes. FISH of the 5S and 45S rDNA probes on L. longiflorum metaphase complements showed overlapping signals at proximal positions of the short arms of chromosomes 4 and 7, a single 5S rDNA signal on the secondary constriction of chromosome 3, and one 45S rDNA signal adjacent to the 5S rDNA signal on the subdistal part of the long arm of chromosome 3. In L. rubellum, we observed co-localisation of the 5S and 45S rDNA sequences on the short arm of chromosomes 2 and 4 and on the long arms of chromosomes 2 and 3, and two adjacent bands on chromosome 12. Silver staining (Ag-NOR) of the nucleoli and NORs in L. longiflorum and L. rubellum yielded a highly variable number of signals in interphase nuclei and only a few faint silver deposits on the NORs of mitotic metaphase chromosomes. In preparations stained with PI and DAPI, we observed both red-and blue-fluorescing bands at different positions on the L. longiflorum and L. rubellum chromosomes. The red-fluorescing or so-called reverse PI-DAPI bands always coincided with rDNA sites, whereas the blue-fluorescing DAPI bands corresponded to C-bands. Based on these techniques, we could identify most of chromosomes of the L. longiflorum and L. rubellum karyotypes.
Genome, 2001
Detailed karyotypes of Lilium longiflorum and L. rubellum were constructed on the basis of chromosome arm lengths, C-banding, AgNO 3 staining, and PI-DAPI banding, together with fluorescence in situ hybridisation (FISH) with the 5S and 45S rDNA sequences as probes. The C-banding patterns that were obtained with the standard BSG technique revealed only few minor bands on heterologous positions of the L. longiflorum and L. rubellum chromosomes. FISH of the 5S and 45S rDNA probes on L. longiflorum metaphase complements showed overlapping signals at proximal positions of the short arms of chromosomes 4 and 7, a single 5S rDNA signal on the secondary constriction of chromosome 3, and one 45S rDNA signal adjacent to the 5S rDNA signal on the subdistal part of the long arm of chromosome 3. In L. rubellum, we observed co-localisation of the 5S and 45S rDNA sequences on the short arm of chromosomes 2 and 4 and on the long arms of chromosomes 2 and 3, and two adjacent bands on chromosome 12. Silver staining (Ag-NOR) of the nucleoli and NORs in L. longiflorum and L. rubellum yielded a highly variable number of signals in interphase nuclei and only a few faint silver deposits on the NORs of mitotic metaphase chromosomes. In preparations stained with PI and DAPI, we observed both red-and blue-fluorescing bands at different positions on the L. longiflorum and L. rubellum chromosomes. The red-fluorescing or so-called reverse PI-DAPI bands always coincided with rDNA sites, whereas the blue-fluorescing DAPI bands corresponded to C-bands. Based on these techniques, we could identify most of chromosomes of the L. longiflorum and L. rubellum karyotypes.
Chromosome Research, 2000
Interspecific hybrids between Lilium longiflorum (L, 2n=2x=24) andLilium rubellum (R, 2n=2x=24) were produced with the aim of transferring desirable horticultural traits from L. rubellum to L. longiflorum. All F1 hybrids (LR, 2n=2x=24) and BC1 individuals (LLR, 2n=3x=36) were phenotypically uniform for plant height, flowering time, leaf shape and flower colour. The BC1 plants were, in spite of their triploid nature, fertile and could be used as a female parent in backcrossings with autotetraploid L. longiflorum (LLLL, 2n=4x=48). Twelve BC2 individuals were obtained and three of them were selected for further chromosome analysis. As L. longiflorum and L. rubellum chromosomes were indistinguishable in the hybrids, genomic in- situ hybridization (GISH) was applied to establish the parentage of the chromosomes of the F1 hybrids and the BC1 and BC2 progenies. GISH confirmed the LLRR constitution of the doubled amphimonoploid (allodiploid), and the LLR constitution of all BC1 plants. The three selected BC2 plants were, as expected, aneuploid, containing three complete sets of L. longiflorum chromosomes and six, seven or eight L. rubellum chromosomes, respectively. However, L/R translocation or recombinant chromosomes could not be demonstrated in the mitotic metaphase complements of the F1, BC1 and BC2 plants. In spite of the high frequencies of homoeologous recombination in the F1 hybrids (LR) pollen was found to be sterile in all cases. At metaphase I of the pollen mother cells of the BC1 plants, genome painting did not reveal any cases of homoeologous pairing and recombination between L and R chromosomes. This lack of exchange between homoeologous chromosome segments indicates complete preferential pairing of the L and R chromosomes in the F1 (amphidiploid) and BC1 plants. It seems that the preferential pairing in the F1 and BC1 hybrids hinder the introgression of the chromosome segments or species-specific genes into the recipient for breeding purposes.
Chromosomal differentiation and genome size in three European mountain Lilium species
Plant Systematics and Evolution, 2003
Three related and taxonomically close species of the genus Lilium (L. pyrenaicum Gouan, L. pomponium L. and L. carniolicum Bernh.), all of them with 2n=24 chromosomes, have been studied for chromosomal differentiation, using fluorochrome banding and fluorescence in situhybridization (FISH), and for genome size and GC percentage using flow cytometry. The total DNA content of L. pomponium (2C=70.26 pg) was about 5% higher than that of L. pyrenaicum (2C=67.74) and L. carniolicum (2C=67.37 pg), while GC percentage was higher in this last species (36.60%) than in L. pomponium (35.56%) and lower than in L. pyrenaicum (37.92%). Silver staining, fluorochrome banding with chromomycin A3 (CMA) and fluorescence in situ hybridization (FISH) clearly pointed out the number of nucleoli, the number and position of GC-rich bands and the number and location of rDNA sites thus permitting distinction of the three species at chromosomal level. Two families of ribosomal genes, 18S-5.8S-26S (18S) and 5S rRNA genes, were separated onto different pairs in chromosome complements of examined species. Chromosome regions containing both kinds of rRNA genes were also GC-rich regions. The results revealed a clear interspecific differentiation at the chromosomal level and permitted the discussion about relationships among the species.
Development of genomic resources for ornamental lilies (Lilium L.)
2012
Lily (Lilium L.) is a perennial bulbous ornamental, belonging to subclass Monocotyledonae and family Liliaceae. Lily, according to statistics of Dutch auctions, is the fifth most important cut flower and the second in flower bulbs based on acreage. This species has been extensively used for cytogenetic studies, but molecular genetic studies are limited. The heterogenic nature and the very complex and huge genome (36 Gb) of lily might be the reason for this. To improve the efficiency of breeding and selection in this species, and set up the basis for genetic studies in Lilium, genomic resources are needed. Next generation sequencing (NGS) technology (454 pyro-sequencing) was used to sequence the transcriptomes (RNA-seq) of four lily cultivars: ‘Connecticut King’, ‘White Fox’, ‘Star Gazer’, and Trumpet that belong to the four most important hybrid groups: Asiatic, Longiflorum, Oriental, and Trumpet respectively. Successfully, 52,172 unigenes with an average length of 555 bp were devel...
Repetitive DNA sequences accelerate molecular cytogenetic research in plants with small chromosomes
Indonesian Journal of Biotechnology, 2019
Repetitive DNA sequences are highly abundant in plant genomes and are favorable probes for chromosome identification in plants. However, it is difficult to conduct studies on the details of metaphase chromosome structures in plants with small chromosomes due to their highly condensed status. Therefore, identification of homologous chromosomes for karyotyping and analyzing chromosome structures is a challenging issue for cytogeneticists without specific probes and precise chromosome stages. In this study, five repetitive DNA probes, i.e., 5S and 45S ribosomal DNAs (rDNAs), melon centromeric sequence (Cmcent), cucumber subtelomeric sequence (Type I), and microsatellite (CT)10 repeats, were used to identify primary constrictions and homologous chromosomes for karyotyping. Four and two loci of 45S rDNA were respectively observed on metaphase and pachytene chromosomes of Abelia × grandiflora. Cmcent was detected on both primary constrictions of melon pachytene and metaphase chromosomes. ...
Development of expressed sequence tag derived-simple sequence repeats in the genus Lilium
Genes & Genomics, 2011
Although lily is the second largest flower crop in cutting flower commodity, only six simple sequence repeats SSRs have been reported. Thus, we developed expressed sequence tag derived-SSRs (EST-SSRs) for the Lilium genus. Among 2,235 unique ESTs, 754 ESTs contained SSR motifs, among which 165 ESTs were amenable to primer design. Among these 165 EST-SSRs, 131 EST-SSRs showed amplification in at least one Lilium species, and 76 EST-SSRs showed amplification in at least nine species. Of the 76 EST-SSRs, 47 showed amplification in all Lilium species analyzed. Using 10 breeding lines, we selected 19 EST-SSRs that had the highest number of alleles and polymorphism information content. The polymorphism information content values of these selected EST-SSRs ranged from 0.49 to 0.94 with an average of 0.76, which are higher than other plant species. The phylogenetic dendrogram derived from the amplification profiles of the 19 high polymorphic EST-SSRs was congruent with the genetic background of the 84 selected lily accessions and hybrids, which are available in commerce. Thus, the developed EST-SSRs will be very useful in germplasm management, genetic diversity analysis, cultivar finger printing, and molecular breeding in the lily.
Construction of a DNA library from chromosome 4 of rice (Oryza sativa) by microdissection
Cell Research, 1998
A simple method to create a chromosome-specific DNA library of rice, including microdissection, amplification, characterization and cloning, is described. Rice chromosome 4 from a metaphase cell has been isolated and amplified by the Linker Adapter PCR (LA-PCR). The PCR products were labeled as probes with DIG-11-dUTP using the random priming method. Southern blot analysis with rice genomic DNA and specific RFLP markers demonstrated that the PCR products were derived from rice chromosome 4. A large library comprising over 100,000 recombinant plasmid microclones from rice chromosome 4 was constructed. Colony hybridization showed that 58% of the clones contained single or low-copy sequences and 42% contained repetitive sequences. The size of inserts generated by PCR ranged from 140bp to 500bp.This method will facilitate cloning of the specific chromosome DNA markers and important genes of rice.
Chinese Science Bulletin, 1998
A method for microdissection, isolation and amplification of plant chromosomal fragments using laser microbeam and a glass microneedle was established. Firstly, 7H chromosome of barley (Hordeum vulgnre L. ) was dissected by Nd: YAG laserbeam with suitable parameters and the fragment comprising a satellite was isolated with a glass microneedle which was fixed on a micromanipulator. Then, the chromosomal fragment DNA was amplified by LA-PCR (linker adaptor PCR) for two rounds. The size of the DNA fragments of PCR products varied from 500-3 000 bp and the PCR products originated from the genome of barley were verified by Southern hybridization. Compared with previous reports, there are some advantages in this research. The performance is easier. the dissection is more precise and the cost is low. It also permits efficient amplification with only one single chromasome fragment. Laser micmbeam-glass microneedle method may be useful in the microdissection of special chromosome regions, especially in plants with middle or small chromosomes.