FISH Karyotype and GISH Meiotic Pairing Analyses of a Stable Intergeneric Hybrid xBrassicoraphanus Line BB#5

Abstract

xBrassicoraphanus line BB#5, a new synthetic intergeneric hybrid between Brassica rapa L. ssp. pekinensis and Raphanus sativus L. var. rafiphera induced by N-methyl-N-nitroso-urethane mutagenesis in microspore culture, shows high seed fertility and morphological uniformity. Dual-color fluorescence in situ hybridization (FISH) using 5S and 45S rDNA probes and genomic in situ hybridization (GISH) using B. rapa genomic DNA probe were carried out to analyze the chromosome composition and the meiosis pairing pattern compared to its parental lines. The somatic chromosome complement is 2n = 38, which consists of 17 metacentric and two submetacentric chromosomes with lengths of 2.18 to 5.01 µm. FISH karyotype analysis showed five and eight pairs of 5S and 45S rDNA loci. GISH meiosis pairing analysis showed that 19 complete bivalents were most frequent and accounted for 42% of the 100 pollen mother cells examined. Based on chromosome number, size, morphology, rDNA distribution, and meiosis pairing pattern, both parental genomes of B. rapa and R. sativus appear to exist in xBrassicoraphanus line BB#5, demonstrating its genome integrity. Such stable chromosome constitutions and meiotic pairing patterns in somatic and meiotic cells are very rare in natural and synthetic intergeneric hybrids. Chromosomal studies and genetic and phenotypic changes in allopolyploids are discussed. The results presented herein will be useful for further genomic study of xBrassicoraphanus lines and their improvement as promising new breeding varieties.

A modified method for preparing meiotic chromosomes based on digesting pollen mother cells in suspension

“…Currently, the heterogeneous PMCs and their daughter cells in many plants, such as some polyploid plants and some interspecific hybrids, have become objects of cytogenetic research [ 18 , 33 ]. Accurate results should be obtained from the analysis of massive numbers of spreads instead of relying on one or a few cells.…”

“…Unfortunately, anther and/or bud walls were not permeated by the enzyme solution and PMCs grouped together. Thus, PMC walls could not be totally eliminated using low concentrations of enzymes and short digestion times [ 18 ]. Although higher concentration enzymes and longer digestion times may eliminate the walls completely, many PMC protoplasts are also degraded.…”

Subgenome Discrimination in Brassica and Raphanus Allopolyploids Using Microsatellites

“…Karyograms were finalized using Adobe Photoshop CC version 25.3, whereas ideograms were generated using Adobe Illustrator CC version 22.4. Homologous chromosomes were identified based on their FISH signals, morphological characteristics, and lengths, considering previous karyotype data for B. rapa, B. oleracea, and B. napus [5], B. nigra and B. juncea [42], and R. sativus and ×Brassicoraphanus [43].…”

Meiotic Chromosome Stability and Suppression of Crossover Between Non-homologous Chromosomes in xBrassicoraphanus, an Intergeneric Allotetraploid Derived From a Cross Between Brassica rapa and Raphanus sativus

“…The fragmented genomic DNA of B. rapa and R. sativus was labeled with digoxigenin-11-dUTP and biotin-16-dUTP (Roche, Germany) by nick translation, respectively. For GISH with A-and R-genome probes in xBrassicoraphanus, the methods of Kwon and Kim (2009) and Belandres et al (2015) were adopted with modifications. First, chromosome spreads were incubated with digoxigenin-and biotin-labeled probes along with Fluorescein Avidin DCS (diluted 1:100) (Vector Laboratories, United States) at 37 • C for 1 h. After washing three times for 10 min each in 4X SSCT, reactions were performed with rhodamine-conjugated sheep anti-digoxigenin antibody (diluted 1:10) (Roche, Germany) and biotinylatedanti-avidin D antibody (diluted 1:100) (Vector Laboratories, United States) at 37 • C for 1 h. In the final reaction, digrhodamine and biotin-avidin labeled probes were detected with anti-sheep Texas Red antibody (diluted 1:100) and Fluorescein Avidin DCS (diluted 1:100) (Vector Laboratories, United States), respectively.…”