Cytogenetic Analyses Revealed Different Genome Rearrangement Footprints in Four ×Brassicoraphanus Lines with Different Fertility Rates

×Brassicoraphanus (AARR, 2n = 38) is a synthetic intergeneric allopolyploid between Brassica rapa L. ssp. pekinensis (AA, 2n = 20) and Raphanus sativus L. var. rafiphera (RR, 2n = 18). Abnormalities in meiosis are main causes for infertility, especially in recent intergeneric allopolyploids. Several ×Brassicoraphanus lines showing varied fertility rates were produced previously, but no cytogenetic data specifying the reasons for infertility have been reported. In this study, we performed cytogenetic analyses in BB4, BB6, BB12, and BB50 lines to evaluate their chromosomal composition and behavior during meiosis. The four lines had relatively small chromosomes, ranging in length from 1.82 to 5.72 μm. BB6, BB12, and BB50 have euploid chromosome number of 2n = 38, whereas BB4 is an aneuploid with 2n ‒ 1 = 37. Fluorescent in situ hybridization karyotype analysis by using 5S/45S rDNA revealed 5/7, 6/7, 5/5 and 5/5 pairs in BB4, BB6, BB12 and BB50, respectively. Genomic in situ hybridization analysis on cells in prophase I revealed varying frequencies of tetravalent pairing and sticky, ring, rod, and laggard chromosomes across the lines, which were more abundant in BB4 and BB6. Unlike BB4 and BB6, both BB12 and BB50 are known to have relatively higher seed fertility and uniform plant morphology. The varied degrees of chromosomal pairing stability during meiosis could explain the different fertility rates among the four ×Brassicoraphanus lines in this study. These data might facilitate breeding programs of ×Brassicoraphanus and further cytogenomic analyses.