Multicolor fluorescence in situ hybridization with combinatorial labeling probes enables a detailed karyotype analysis of Larix principis-rupprechtii

Bo Liu,Wenqin Song,Chen Ruiyang,Qi Liwang

doi:10.4067/s0716-97602007000100003

Abstract

The chromosomes (2n = 2x = 24) of Larix principis-rupprechtii are composed of six pairs of large metacentrics and six pairs of medium-sized submetacentrics. The identification of homologous pairs is hampered by their high degree of similarity at the morphological level in each group. As one of the most extensively used methods in molecular cytogenetics producing chromosome landmarks, fluorescence in situ hybridization (FISH) has significantly facilitated karyotype construction, especially in species with morphologically similar chromosomes. This study developed a simple but effective use of combinatorial labeling probes to distinguish chromosomes of Larix principis-rupprechtii by multicolor FISH. Three highly repetitive sequences in Larix were selected: 25S rDNA hybridized at all of the secondary constrictions of two pairs of metacentrics and the largest pair of submetacentrics; 5S rDNA hybridized at subtelomeric sites of one pair of metacentrics that also harboured 25S rDNA on different arms; LPD family sequences are tandem repeats hybridized at proximal regions of 22 chromosomes. The three different probes were labeled with only two different labels, hybridized to metaphase chromosomes of Larix principis-rupprechtii, simultaneously visualized, and unequivocally distinguished in a single FISH experiment. These multicolor FISH marks largely improved the karyotype analysis of Larix principis-rupprechtii.

Highlights

The chromosomes (2n = 2x = 24) of Larix principis-rupprechtii are composed of six pairs of large metacentrics and six pairs of medium-sized submetacentrics
Combinatorial labeling of probes has scarcely been used for molecular cytogenetics of gymnosperms
We developed a simple and efficient technique of multicolor fluorescence in situ hybridization in Larix principis-rupprechtii, which allows the use of probes labeled with two standard labels to map simultaneously three different DNA sequences along somatic chromosomes

Summary

Introduction

Key terms: combinatorial labeling probes, fluorescence in situ hybridization, karyotype, Larix principisrupprechtii, LPD repeats, 25S and 5S rDNA We developed a simple and efficient technique of multicolor fluorescence in situ hybridization in Larix principis-rupprechtii, which allows the use of probes labeled with two standard labels to map simultaneously three different DNA sequences along somatic chromosomes.

Results

Conclusion